Bicyclic compounds capable of inhibiting tyrosine kinases of the epidermal growth factor receptor family

ABSTRACT

Described are heterocyclic pyrimidine derivatives and a method of inhibiting epidermal growth factor by treating, with an effective inhibiting amount, a mammal, in need thereof, a compound of the formula:  
                 
 
     where:  
     at least two of A-E are nitrogen, with the remaining atom(s) carbon;  
     X═NH or NR 7 , such that R 7 ═lower alkyl (1-4 carbon atoms), OH, NH 2 , lower alkoxy (1-4 carbon atoms) or lower monoalkylamino (1-4 carbon atoms);  
     n=0, 1, 2.  
     The remaining substituents are described in detail in the specification.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/183,190, filed Oct. 30, 1998 now U.S. Pat. No. ______, which is acontinuation of U.S. application Ser. No. 08/811,797, filed Mar. 6,1997, now U.S. Pat. No. 6,084,095, issued Jul. 4, 2000, which is adivisional application of U.S. Ser. No. 08/358,351, filed Dec. 23, 1994,now U.S. Pat. No. 5,654,307, which is a continuation-in-part of U.S.application Ser. No. 08/186,735, filed Jan. 25, 1994, now abandoned anda continuation-in-part of U.S. application Ser. No. 186,745, filed Jan.24, 1994, now abandoned, all of which are hereby incorporated byreference.

TECHNICAL FIELD

[0002] The present invention relates to bicyclic heteroaromaticcompounds which inhibit the epidermal growth factor receptor and relatedreceptors and, in particular, their tyrosine kinase enzymic activity.

BACKGROUND ART

[0003] Cancer is generally a disease of the intracellular signallingsystem, or signal transduction mechanism. Cells receive instructionsfrom many extracellular sources, instructing them to either proliferateor not to proliferate. The purpose of the signal transduction system isto receive these and other signals at the cell surface, get them intothe cell, and then pass the signals on to the nucleus, the cytoskeleton,and transport and protein synthesis machinery. The most common cause ofcancer is a series of defects, either in these proteins, when they aremutated, or in the regulation of the quantity of the protein in the cellsuch that it is over or under produced. Most often, there are keylesions in the cell which lead to a constitutive state whereby the cellnucleus receives a signal to proliferate, when this signal is notactually present. This can occur through a variety of mechanisms.Sometimes the cell may start to produce an authentic growth factor forits own receptors when it should not, the so-called autocrine loopmechanism. Mutations to the cell surface receptors, which usually signalinto the cell by means of tyrosine kinases, can lead to activation ofthe kinase in the absence of ligand, and passing of a signal which isnot really there. Alternatively, many surface kinases can beoverexpressed on the cell surface leading to an inappropriately strongresponse to a weak signal. There are many levels inside the cell atwhich mutation or overexpression can lead to the same spurious signalarising in the cell, and there are many other kinds of signalling defectinvolved in cancer. This invention touches upon cancers which are drivenby the three mechanisms just described, and which involve cell surfacereceptors of the epidermal growth factor receptor tyrosine kinase family(EGFR). This family consists of the EGF receptor (also known as Erb-B1),the Erb-B2 receptor, and its constituitively active oncoprotein mutantNeu, the Erb-B3 receptor and the Erb-B4 receptor. Additionally, otherbiological processes driven through members of the EGF family ofreceptors can also be treated by compounds of the invention describedbelow.

[0004] The EGFR has as its two most important ligands Epidermal GrowthFactor (EGF) and Transforming Growth Factor alpha (TGFalpha). Thereceptors appear to have only minor functions in adult humans, but areapparently implicated in the-disease process of a large portion of allcancers, especially colon and breast cancer. The closely related Erb-B2Erb-B3 and Erb-B4 receptors have a family of Heregulins as their majorligands, and receptor overexpression and mutation have beenunequivocally demonstrated as the major risk factor in poor prognosisbreast cancer. Additionally, it has been demonstrated that all four ofthe members of this family of receptors can form heterodimericsignalling complexes with other members of the family, and that this canlead to synergistic transforming capacity if more than one member of thefamily is overexpressed in a malignancy. Overexpression of more than onefamily member has been shown to be relatively common in humanmalignancies.

[0005] The proliferative skin disease psoriasis has no good cure atpresent. It is often treated by anticancer agents such as methotrexate,which have very serious side effects, and which are not very effectiveat the toxicity-limited doses which have to be used. It is believed thatTGFalpha is the major growth factor overproduced in psoriasis, since 50%of transgenic mice which overexpress TGF alpha develop psoriasis. Thissuggests that a good inhibitor of EGFR signalling could be used as anantipsoriatic agent, preferably, but not necessarily, by topical dosing.

[0006] EGF is a potent mitogen for renal tubule cells. Fourfoldincreases in both EGF urinary secretion and EGF mRNA have been noted inmice with early stage streptozoicin-induced diabetes. In additionincreased expression of the EGFR has been noted in patients withproliferative glomerulonephritis (Roychaudhury et al. Pathology 1993,25, 327). The compounds of the current invention should be useful intreating both proliferative glomerulonephritis and diabetes-inducedrenal disease.

[0007] Chronic pancreatitis in patients has been reported to correlatewith large increases in expression for both EGFR and TGF alpha. (Korc etal. Gut 1994, 35, 1468). In patients showing a more severe form of thedisease, typified by an enlargement of the head of the pancreas, therewas also shown to be overexpression of the erb-B2 receptor (Friess etal. Ann. Surg. 1994, 220, 183). The compounds of the current inventionshould prove useful in the treatment of pancreatitis.

[0008] In the processes of blastocyte maturation, blastocyteimplantation into the uterine endometrium, and other periimplantationevents, uterine tissues produce EGF and TGF alpha (Taga Nippon SankaFujinka Gakkai Zasshi 1992, 44, 939), have elevated levels of EGFR(Brown et al. Endocrinology, 1989, 124, 2882), and may well be inducedto produce heparin-binding EGF by the proximity of the developing, butnot arrested, blastocyte (Das et al. Development 1994, 120, 1071). Inturn the blastocyte has quite a high level of TGF alpha and EGFRexpression (Adamson Mol. Reprod. Dev. 1990, 27, 16). Surgical removal ofthe submandibular glands, the major site of EGF secretion in the body,and treatment with anti-EGFR monoclonal antibodies both greatly reducefertility in mice (Tsutsumi et al. J. Endocrinology 1993, 138, 437), byreducing successful blastocyte implantation. Therefore, compounds of thecurrent invention should prove to have useful contraceptive properties.

[0009] PCT patent application Nos. WO92/07844 published May 14, 1992 andWO92/14716 published Sep. 3, 1992 describe 2,4-diaminoquinazoline aspotentiators of chemotherapeutic agents in the treatment of cancer.

[0010] PCT published application No. WO92/20642 published Nov. 26, 1992discloses bismono- and bicyclic aryl and heteroaryl compounds whichinhibit EGF and/or PDGF receptor tyrosine kinase.

[0011] It is an object of the present invention to inhibit the mitogeniceffects of epidermal growth factor utilizing an effective amount ofbicyclic pyrimidine derivatives, in particular fused heterocyclicpyrimidine derivatives.

[0012] It is another object of the present invention to describebicyclic pyrimidine derivatives, in particular fused heterocyclicpyrimidine derivatives, as inhibitors of the EGF, Erb-B2 and Erb-B4receptor tyrosine kinases.

[0013] It is yet another object of the present invention to describebicyclic pyrimidine derivatives, in particular fused heterocyclicpyrimidine derivatives, that are useful at low dosages as inhibitors ofEGF-induced mitogenesis. This therefore leads to a further object ofcompounds having extremely low cytotoxicity.

[0014] It is a further object of the present invention to describebicyclic pyrimidine derivatives, in particular fused heterocyclicpyrimidine derivatives, that are useful in suppressing tumors,especially breast cancers, where mitogenesis is heavily driven by EGFRfamily members.

[0015] It is another object of the present invention to describebicyclic pyrimidine derivatives, in particular fused heterocyclicpyrimidine derivatives, that have utility as chronic therapy asinhibitors of EGF-induced responses.

[0016] It is another object of the current invention to describebicyclic pyrimidine derivatives, in particular fused heterocyclicpyrimidine derivatives, that have utility as therapeutic agents againstproliferative overgrowth diseases, including but not limited to,synovial, pannus invasion in arthritis, vascular restenosis, psoriasisand angiogenesis. The compounds disclosed herein also are useful totreat pancreatitis and kidney disease and as a contraceptive agent.

SUMMARY OF THE INVENTION

[0017] Described is a method to inhibit epidermal growth factor bytreating, with an effective inhibiting amount, a mammal, in needthereof, a compound of Formula I:

[0018] wherein at least one, and as many as three of A-E are nitrogen,with the remaining atom(s) carbon, or any two contiguous positions inA-E taken together can be a single heteroatom, N, O or S, in which caseone of the two remaining atoms must be carbon, and the other can beeither carbon or nitrogen;

[0019] X═O, S, NH or NR⁷, such that R⁷═lower alkyl (1-4 carbon atoms),OH, NH₂, lower alkoxy (1-4 carbon atoms) or lower monoalkylamino (1-4carbon atoms);

[0020] n=0, 1, 2;

[0021] R¹═H or lower alkyl (1-4 carbon atoms); if n=2, R¹ can beindependently H or lower alkyl (1-4 carbon atoms) on either linkingcarbon atom;

[0022] R² is lower alkyl (1-4 carbon atoms), cycloalkyl (3-8 carbonatoms), lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms),nitro, halo (fluoro, chloro, bromo, iodo), lower perfluoroalkyl (1-4carbon atoms), hydroxy, lower acyloxy (1-4 carbon atoms; —O—C(O)R),amino, lower mono or dialkylamino (1-4 carbon atoms), lower mono ordicycloalkylamino (3-8 carbon atoms), hydroxymethyl, lower acyl (1-4carbon atoms; —C(O)R), cyano, lower thioalkyl (1-4 carbon atoms),lower-sulfinylalkyl (1-4 carbon atoms), lower sulfonylalkyl (1-4 carbonatoms), thiocycloalkyl (3-8 carbon atoms), sulfinylcycloalkyl (3-8carbon atoms), sulfonylcycloalkyl (3-8 carbon atoms), sulfonamido, lowermono or dialkylsulfonamido (1-4 carbon atoms), mono ordicycloalkylsulfonamido (3-8 carbon atoms), mercapto, carboxy,carboxamido (—C(O)—NH₂), lower mono or dialkylcarboxamido (1-4 carbonatoms), mono or dicycloalkylcarboxamido (3-8 carbon atoms), loweralkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbonatoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbonatoms), lower alkynyl (2-4 carbon atoms), or two R² taken together oncontiguous carbon atoms can form a carbocyclic ring of 5-7 members or amonounsaturated 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino or thiomorpholino ring; and

[0023] m=0-3, wherein Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl,pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl andquinazolinyl;

[0024] R³, R⁴, R⁵ and R⁶ are independently, not present, H, lower alkyl(1-4 carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy (1-4carbon atoms), cycloalkoxy (3-8 carbon atoms), hydroxy, lower acyloxy(1-4 carbon atoms), amino, lower mono or dialkylamino (1-4 carbonatoms), lower mono or dicycloalkylamino (3-8 carbon atoms), lower alkyl(1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms), carbonato(−0C(O)OR) where the R is lower alkyl of 1 to 4 carbon atoms orcycloalkyl of 3-8 carbon atoms;

[0025] or ureido or thioureido or N- or O- linked urethane any one ofwhich is optionally substituted by mono or di-lower alkyl (1-4 carbonatoms) or cycloalkyl (3-8 carbon atoms);

[0026] lower thioalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbonatoms), mercapto, lower alkenyl (2-4 carbon atoms), hydrazino,N′-loweralkylhydrazino (1-4 carbon atoms), lower acylamino (1-4 carbon atoms),hydroxylamino, lower O-alkylhydroxylamino (1-4 carbon atoms);

[0027] or any two of R³-R⁶ taken together on contiguous carbon atoms canform a carbocyclic ring of 5-7 members or a monounsaturated1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl, furanyl,pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl, diazolanyl,piperazinyl, morpholino or thiomorpholino ring;

[0028] any lower alkyl group substituent on any of the substituents inR³-R⁶ which contain such a moiety can be optionally substituted with oneor more of hydroxy, amino, lower monoalkylamino, lower dialkylamino,N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino,N-thiomorpholino or N-piperazino groups;

[0029] if one or more of A through E are N, then any of R³-R⁶ on aneighboring C atom to one of the N atoms, cannot be either OH or SH; and

[0030] if any of the substituents R¹, R², R³, R⁴, R⁵ or R⁶ containchiral centers, or in the case of R¹ create chiral centers on thelinking atoms, then all stereoisomers thereof both separately and asracemic and/or diastereoisomeric mixtures are included.

[0031] Described also is a method to inhibit epidermal growth factor bytreating, with an effective inhibiting amount, a mammal, in needthereof, a compound of Formula II:

[0032] wherein

[0033] Ar, n, m, R₁-R₇ and X are the same as in Formula I;

[0034] R⁸ is alkyl of from 1-4 carbon atoms or amino or mono or diloweralkyl (1-4 carbon atoms) amino.

[0035] The invention is also applicable to the compositions of FormulaeI and II with the proviso that at least one of the R³-R⁶ substituentsmust be taken singly as a substituent other than hydrogen, halo, loweralkyl (1-4 carbon atoms) or lower alkoxy (1-4 carbon atoms), and withthe proviso that A, B, D and E must all be taken singly as carbon ornitrogen atoms.

[0036] The invention is also applicable to the compositions of FormulasI and II with the further provisos that when:

[0037] E and B are nitrogen and D and A are carbon, R³ and R⁶ are notpresent, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹ is H or loweralkyl; Ar is aryl unsubstituted or substituted by halo, amino, hydroxyor amino; or n is 1 or 2, R¹ is H or lower alkyl; and Ar is aheterocyclic ring, then R⁴ cannot be hydrogen amino, lowermonoalkylamino unsubstituted or substituted by hydroxy, lowermonocycloalkylamino unsubstituted or substituted by hydroxy, lowerdialkylamino unsubstituted or substituted by hydroxy, orheterocyclicalkyl amino; or

[0038] E and B are nitrogen and D and A are carbon, R³ and R⁶ are notpresent, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹ is H or loweralkyl; Ar is aryl, then R⁴ cannot be lower alkylthio; or

[0039] E and B are nitrogen and D and A are carbon, R³ and R⁶ are notpresent, R⁵ is hydrogen, X is NH, n is 1, R¹ is H, Ar is phenyl parasubstituted by diethylamino, then R⁴ cannot be, amino; or

[0040] E and B are nitrogen and D and A are carbon, R³ and R⁶ are notpresent, R⁵ is hydrogen, X is NH, n is 1, R¹ is H, Ar is 2-furanyl, thenR⁴ cannot be ethylthio or that at least one of the group R³ and R⁵ or R⁴and R⁵ substituents must be other then hydrogen, lower alkyl (1-4 carbonatoms) or lower alkoxy (1-4 carbon atoms).

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 is an effect of Examples 6 and 7 on EGF receptorautophosphorylation in A431 human epidermoid carcinoma;

[0042]FIG. 2 is an effect of Example 8 on EGF receptorautophosphorylation in A431 human epidermoid carcinoma;

[0043]FIG. 3 is a time course for the inhibition of EGF receptorautophosphorylation in A431 by Example 27;

[0044]FIG. 4 is an effect of Example 27 on EGF receptorautophosphorylation in A431 cells;

[0045]FIG. 5 is an inhibition of EGF receptor autophosphorylation inA431 human epidermoid carcinoma by Example 40;

[0046]FIG. 6 is an effect of Example 40 on growth factor-mediatedtyrosine phosphorylation in Swiss 3T3;

[0047] As FIG. 7 is an effect of Example 40 on growth factor dependentexpression or c-jun mRNA in Swiss 3T3 mouse fibroblasts;

[0048]FIG. 8 is an effect of Example 40 on growth factor mediatedexpression of p39^(c-jun);

[0049]FIG. 9 is an effect of Example 59 of EGF receptorautophosphorylation in A431 human epidermoid carcinoma;

[0050]FIG. 10 is an effect of Example 60 on EGF receptorautophosphorylation in A431 human epidermoid carcinoma;

[0051]FIG. 11 is an effect of Example 61 on EGF receptorautophosphorylation in A431 human epidermoid carcinoma;

[0052]FIG. 12 is an effect of Example 70 on EGF receptorautophosphorylation in A431 human epidermoid carcinoma;

[0053]FIG. 13 is a chart showing an inhibition of EGF receptor tyrosinekinase by Example 27;

[0054]FIG. 14 is a graph showing an effect of Example 40 on growthfactor-mediated mitogenesis in Swiss 3T3 murine fibroblasts;

[0055]FIG. 15 is a photograph of an NIH 3T3 mouse fibroblast line,transfected with the human EGFR gene showing a normal flattenedmorphology;

[0056]FIG. 16 is a photograph of the same cell line treated with 100ng/mL of EGF showing a typical spindly transformed morphology; and

[0057]FIG. 17 is a photograph of the same cell line in the presence ofboth 100 ng/mL of EGF and 5 μm of Example 27 showing the morphologyreverted from the transformed type back to the normal type.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0058] 1. A preferred form of the invention has X═NH, n=0 or 1, in whichcase R¹═H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one lower alkoxyor halogen.

[0059] 2. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ or R⁴ H, with the other one amino.

[0060] 3. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ or R⁴ H, with the other one lower monoor dialkylamino.

[0061] 4. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ or R⁴ H, with the other one hydrazino.

[0062] 5. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ or R⁴ H, with the other one loweralkyl.

[0063] 6. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ and R⁴ lower alkoxy.

[0064] 7. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon; with A nitrogen and R³ and R⁴ lower alkyl.

[0065] 8. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen, and R³ or R⁴ amino, with the other one loweralkoxy.

[0066] 9. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen, and R³ or R⁴ lower mono or dialkylamino, withthe other one lower alkoxy.

[0067] 10. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, B,D & E carbon, with A nitrogen and R³ lower mono or dialkylamino, with R⁴hydroxy.

[0068] A suitable ring structure for groups 1-10 is:

[0069] 11. Another preferred form of the invention has X═NH; n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, B,D & E carbon, with A nitrogen, and R³ and R⁴ taken together aredioxymethylene, dioxyethylene, 2,3-fused piperazine, 2,3-fusedmorpholine or 2,3-fused thiomorpholine. Suitable ring structures are:

[0070] 12. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,D & E carbon, with B nitrogen and R⁴ lower alkoxy or halogen.

[0071] 13. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,D & E carbon, with B nitrogen and R⁴ amino.

[0072] 14. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,D & E carbon, with B nitrogen and R⁴ lower mono or dialkylamino.

[0073] 15. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,D & E carbon, with B nitrogen and R⁴ hydrazino.

[0074] 16. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,C & E carbon, with B nitrogen and R⁴ lower alkyl.

[0075] A suitable ring structure for groups 12-16 is:

[0076] 17. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ lower alkoxy or halogen.

[0077] 18. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ amino.

[0078] 19. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ lower mono or dialkylamino.

[0079] 20. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ hydrazino.

[0080] 21. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ lower alkyl.

[0081] A suitable ring structure for groups 17-21 is:

[0082] 22. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other one loweralkoxy.

[0083] 23. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other one amino.

[0084] 24. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other one lowermono or dialkylamino.

[0085] 25. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other onehydrazino.

[0086] 26. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other one loweralkyl.

[0087] 27. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ and R⁴ lower alkoxy.

[0088] 28. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ and R⁴ lower alkyl.

[0089] 29. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen, and R³ or R⁴ amino, with the other onelower alkoxy.

[0090] 30. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen, and R³ or R⁴ lower mono or dialkylamino,with the other one lower alkoxy.

[0091] 31. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R⁴ lower mono or dialkylamino, with R³hydroxy.

[0092] A suitable ring structure for groups 22-31 is:

[0093] 32. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen, and R³ and R⁴ taken together aredioxymethylene, dioxyethylene, 2,3-fused piperazine, 2,3-fusedmorpholine or 2,3-fused thiomorpholine.

[0094] 33. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, A & D carbon, with B and Enitrogen and R⁴ lower alkoxy.

[0095] 34. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, A & D carbon, with B and Enitrogen and R⁴ lower mono or dialkylamino.

[0096] 35. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, A & D carbon, with B and Enitrogen and R⁴ amino.

[0097] 36. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, A & D carbon, with B and Enitrogen and R⁴ hydrazino.

[0098] A suitable ring structure for groups 33-36 is:

[0099] 37. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, B & D carbon, with A and Enitrogen and R³ and R⁴ lower alkoxy.

[0100] 38. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, B & D carbon, with A and Enitrogen and R³ and R⁴ lower mono or dialkylamino.

[0101] 39. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, B & D carbon, with A and Enitrogen and R³ or R⁴ lower alkoxy, with the other lower mono ordialkylamino.

[0102] 40. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, B & D carbon, with A and Enitrogen and R³ and R⁴ taken together are ethylenedioxy, 2,3-fusedpiperazine, 2,3-fused morpholine or 2,3-fused thiomorpholine.

[0103] A suitable ring structure for groups 37-40 is:

[0104] 41. Another, preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a sulfur atom, with D & E carbon, or A& B are carbon with D and E taken together as a sulfur atom, with R⁴ orR³ H, lower alkyl, lower alkoxy, amino, or lower mono or dialkylamino.

[0105] 42. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are an oxygen atom, with D & E carbon, orA & B are carbon with D and E taken together as an oxygen atom, with R⁴or R³ H, lower alkyl, lower alkoxy, amino, or lower mono ordialkylamino.

[0106] 43. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a nitrogen atom, with D & E carbon, orA & B are carbon with D and E taken together as a nitrogen atom, with R⁴or R³ H, lower alkyl, lower alkoxy, amino, or lower mono ordialkylamino.

[0107] 44. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a sulfur atom with D carbon and Enitrogen, or D and E taken together are a sulfur atom, and A is nitrogenand B is carbon, with R^(¾) H, lower alkyl, lower alkoxy, amino, orlower mono or dialkylamino.

[0108] 45. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are an oxygen atom with D carbon and Enitrogen, or D and E taken together are an oxygen atom, and A isnitrogen and B is carbon, with R^(¾) H, lower alkyl, lower alkoxy,amino, or lower mono or dialkylamino.

[0109] 46. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, Aand B taken together are a nitrogen atom, and D is carbon and E isnitrogen, with R^({fraction (3/6)}). H, or lower alkyl, and R⁴ H, loweralkyl, lower alkoxy, amino, or lower mono or dialkylamino.

[0110] 47. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are an oxygen atom with D nitrogen and Ecarbon, or A and B taken together are a carbon atom with D nitrogen andE oxygen, with R^({fraction (3/6)}) H, lower alkyl, lower alkoxy, amino,or lower mono or dialkylamino.

[0111] 48. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a sulfur atom with D nitrogen and Ecarbon, or A and B taken together are a carbon atom with D nitrogen andE sulfur, with R^({fraction (3/6)}) H, lower alkyl, lower alkoxy, amino,or lower mono or dialkylamino.

[0112] 49. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a nitrogen atom with D nitrogen and Ecarbon, or A and B taken together are a carbon atom with D and Enitrogen atoms, with R^({fraction (3/6)}) H or lower alkyl if onnitrogen, or H, lower alkyl, lower alkoxy, amino, or lower mono ordialkylamino if on carbon.

[0113] Other suitable ring structures are:

[0114] Where Z═nitrogen, oxygen or sulfur

[0115] The compounds of the present invention are prepared according toa number of alternative reaction sequences.

Preparative Routes to Compounds of the Invention

[0116] Scheme 1—Route for Preferred Groups 1-5, R⁴═H

[0117] Displacement of the 2-chloro of 2,6-dichloro-3-nitropyridine iscarried out by cuprous cyanide in NMP. Displacement of the secondchlorine of this nitrile by fluoride at this step can be advantageous.This is followed by a mild reduction of the nitro group, underconditions where the halogen is not hydrogenolysed. Hydrolysis of thenitrile followed by orthoformate cyclization, and Vilsmeier-typechlorination will give the dihalo-pyridopyrimidine. Displacement of themore reactive 4-chlorine with an appropriate amine is followed bydisplacement of the 6-halogen with the appropriate nucleophile, ammonia,lower alkylamine, hydrazine, methoxide, to form the final products. (NMPis a solvent, N-methyl-2-pyrrolidone).

[0118] 1. A preferred form of the invention has X═NH, n=0 or 1, in whichcase R¹═H, the aromatic ring phenyl optionally substituted, B, D & Ecarbon, with A nitrogen and R³ or R⁴ H, with the other one lower alkoxyor halogen.

[0119] 2. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ or R⁴ H, with the other one amino.

[0120] 3. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ or R⁴ H, with the other one lower monoor dialkylamino.

[0121] 4. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ or R⁴ H, with the other one hydrazino.

[0122] 5. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ or R⁴ H, with the other one loweralkyl.

[0123] Scheme 2—Route to Preferred Groups 1-5, R³═H

[0124] Displacement of chlorine from 2-chloro-3,5-dinitropyridine isaccomplished with CuCN in NMP. Reduction of the nitro groups to aminesis followed by hydrolysis of the nitrile to an amide. This is cyclizedto the pyrimidone with orthoformate, which is converted to the chlorideby POCl₃ or possibly turned into the thiomethyl derivative by treatmentwith phosphorus pentasulfide followed by MeI and a mild base.Displacement with the appropriate amine gives the desired 7-aminocompound. The amine functionality can be reductively alkylated oractivated by diazotisation of the amino group under acidic or basicconditions, followed by a reduction to the hydrazide, or conversion intoa lower alkyl ether, or to a halogen followed by a cuprate or Stillecoupling by methods familiar to those skilled in the art. Alternatively,the amine can be reductively aminated, or acylated and reduced to formthe alkylamino side chain.

[0125] Scheme 3—Route to Preferred Groups 6 and 8-10 where R⁴═RO

[0126] The known metalation of 2,6-difluoropyridine is exploited twice.LDA treatment followed by a borate/hydrogen peroxide introduces the3-hydroxy substituent. If the pyridine undergoes the 2nd metalation atthe 4 position, the alcohol can be protected as a TIPS (triisopropylsilyl) ether, which will force the second metalation to the 5-position.Alternative nitrations may be used, such as converting the lithiumintermediate to a stannane and treatment with tetranitromethane, or theuse of NO₂BF₄ (nitronium tetrafluoroborate). The C₁ displacement may beeffected by cuprous cyanide or other sources of cyanide ion. Afternitrile hydrolysis and nitro group reduction, ethyl orthoformate may beused instead of formamide for the cyclization, and it may be that somecyclizations will require displacement of F by MeS prior to thereaction. The 4-position is activated by chlorination, and the sidechainamine is then introduced. The final displacement can be by alkoxide oramine nucleophiles to generate the various dialkoxy and amino-alkoxyspecies, and the appropriate use of R can allow the 7-hydroxyl group tobe unmasked at the end of the synthesis. (LDA means lithium diisopropylamide).

[0127] 6. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ and R⁴ lower alkoxy. 8. Anotherpreferred form of the invention has X═NH, n=0 or 1, in which case R¹═H,the aromatic ring phenyl optionally substituted, B, D & E carbon, with Anitrogen, and R³ or R⁴ amino, with the other one lower alkoxy.

[0128] 9. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen, and R³ or R⁴ lower mono or dialkylamino, withthe other one lower alkoxy.

[0129] 10. Another Preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, B,D & E carbon, with A nitrogen and R³ lower mono or dialkylamino, with R⁴hydroxy.

[0130] Scheme 4—Route to Preferred Group 7

[0131] Use of the 6-alkylquinaldic acid followed by ionic brominationunder forcing conditions gives an anhydride, which is opened withammonia, recyclized to the imide, and then the Hoffman degradationoccurs at the less active carbonyl. Cyclization and ring side chainaddition in the normal manner is followed by a Stille coupling tointroduce the R⁴ alkyl group. At this step alkenyl or aryl substituentscould also be introduced using this coupling technology.

[0132] 7. Another preferred form of the invention has X═NH, n=0 or 1, inwhich case R¹═H, the aromatic ring phenyl optionally substituted, B, D &E carbon, with A nitrogen and R³ and R⁴ lower alkyl.

[0133] Scheme 5—Route to Preferred Groups 8, 9, R³═OR

[0134] Dinitration of 2,6-dihydroxypyridine is followed by conversion tothe very reactive dichlorocompound. The dinitrodichloropyridine issingly displaced by cuprous cyanide in NMP, and then the compound isreduced under mild conditions to the diamine. The nitrile is hydrolysedto the amide, which can then be cyclized to the pyridopyrimidone, whichis 4-chlorinated in the usual fashion. Displacement of the more reactivechlorine with the 4sidechain is followed by displacement of the6-chlorine with alkoxid. For group 9, the amine should be alkylatedappropriately by methods familiar to one skilled in the art.

[0135] Scheme 6—Route to Preferred Group 11

[0136] Compounds of preferred group 11 are specialized cases ofpreferred groups 6, 8, 9 and 10, where R³ and R⁴ are cyclized together.They can be made using the same routes as those described for thepreferred groups, with minor modifications, which will be obvious to oneskilled in the art. For example vicinally substituted alkoxy aminocompounds can be dealkylated, and the corresponding vicinalaminoalcohols can be bisalkylated with an appropriate dihaloalkane.

[0137] 11. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, B,D & E carbon, with A nitrogen, and R³ and R⁴ taken together aredioxymethylene, dioxyethylene, 2,3-fused piperazine, 2,3-Fusedmorpholine or 2,3-fused thiomorpholine.

[0138] Scheme 7—Route for Preferred Groups 12-16

[0139] 2,4-Diamino-5-cyanopyridine can be cyclized directly to many4-benzylaminopyridopyrimidine derivatives by treatment with thebenzylamine and formic acid at high temperature. For less nucleophilicamines 2,4-diamino-5-cyanopyridine is converted via ethylorthoformate/acetic anhydride treatment, followed by cyclization withhydrosulfide ion in anhydrous conditions, to give7-amino-4-thiono-3H-pyrido[4,3-d]pyrimidine. S-Alkylation anddisplacement with an appropriate amine gives the desired product. If R⁴is not amino, the amine can be acylated, or reductively alkylated.Alternatively 2,4-diamino-5-cyanopyridine can be hydrolysed to thecorresponding amide, and this species can be cyclized to7-amino-4-oxo-3H-pyrido[4,3-d]pyrimidine with orthoformate.Diazotization of the 7-amine and replacement with fluorine allows forintroduction of other amine and alkoxide nucleophiles at the end of thesynthesis after the C4 substituent has been introduced in the usualmanner. Diazotization and replacement of the amine with bromide allowsfor Stille couplings at the 7-position.

[0140] 12. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,D & E carbon, with B nitrogen and R⁴ lower alkoxy or halogen.

[0141] 13. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,D & E carbon, with B nitrogen and R⁴ amino.

[0142] 14. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,D & E carbon, with B nitrogen and R⁴ lower mono or dialkylamino.

[0143] 15. Another preferred form of the invention has X═NH, n=0 or 1;in which case R¹═H, the aromatic ring phenyl optionally substituted, A,D & E carbon, with B nitrogen and R⁴ hydrazino.

[0144] 16. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,C & E carbon, with B nitrogen and R⁴ lower alkyl.

[0145] Scheme 8—Route for Preferred Groups 17-21

[0146] 2-Chloro-5-nitropyridine is converted to the corresponding2-fluorocompound by KF in DMSO. Reduction of the nitro group followed bytreatment with Boc anhydride gives the Bocamino derivative, which can bemetalated and carboxylated at the 4-position. Removal of the Boc withTFA and cyclization of the pyrimidone ring with formamide gives6-fluoro-4-oxo-3H-pyrido[3,4-d]pyrimidine. This is 4-chlorinated in theusual manner and the 4-sidechain is introduced via displacement with anappropriate amine. Displacement of the 6-fluorine with appropriatenucleophiles leads to various different final products. If the fluorineis displaced by thiomethoxide, that in turn can be displaced by alkylgroups in Ni-catalyzed Grignard displacements.

[0147] 17. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ lower alkoxy or halogen.

[0148] 18. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ amino.

[0149] 19. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ lower mono or dialkylamino.

[0150] 20. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ hydrazino.

[0151] 21. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & E carbon, with D nitrogen and R³ lower alkyl.

[0152] Scheme 9—Route to Preferred Groups 22-26, R⁴═H

[0153] Nitration of 2-methoxynicotinic acid is followed by displacementof the activated methoxy group and cyclization of the pyrimidone ring,possibly all in one step with formamidine, or alternatively in two stepswith ammonia followed by cyclization with a formamide equivalent. Thecarbonyl is converted to the chloride and displaced with the sidechainin the usual fashion, and the nitro group is then selectively reduced toamino. This can be alkylated, acylated or diazotized. The diazo compoundcan be converted to hydroxy or to the bromide or iodide, and theselatter can undergo a Stille coupling to introduce lower alkyl, alkenyl,aryl, etc. at R³.

[0154] 22. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other one loweralkoxy.

[0155] 23. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other one amino.

[0156] 24. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other one lowermono or dialkylamino.

[0157] 25. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other onehydrazino.

[0158] 26. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ or R⁴ H, with the other one loweralkyl.

[0159] Scheme 10—Route to Preferred Groups 22-26, R³═H

[0160] This route uses the known metalation and carboxylation of2,6-difluoropyridine, followed by displacement of the 2-fluorosubstituent. Cyclization of the pyrimidone ring with formamide, followedby conversion of the carbonyl into chloride in a normal manner gives achlorofluoropyridopyrimidine. The ar(alk)ylamino sidechain is introducedby displacement of the more reactive pyrimidine chlorine, and the R⁴substituent is then introduced by fluoride displacement. Theintroduction of alkyl utilizes displacement of by alkoxide, later ethercleavage to the pyridone, O-triflation and Stille coupling.

[0161] Scheme 11—Route to Preferred Groups 27 and 29-31, R³═RO

[0162] This scheme relies on the metalation of 2,6-difluoropyridinesimilarly to scheme 10. The first metalation is used to introduceoxygen, and the second to introduce the carboxylic acid. If required toforce the second metalation to the 5-position the oxygen may beprotected as the very bulky TIPS ether, and stronger bases than LDA maybe required. Ammonia is introduced at the 2-position under hightemperature and pressure, and the pyridone ring is cyclized, andactivated at the 4-position in the usual manner and then displaced withthe 4-position sidechain. Displacement of the 7-fluoro substituent withan appropriate nucleophile, followed by conversions as described inprevious schemes finishes the synthesis.

[0163] 27. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ and R⁴ lower alkoxy.

[0164] 29. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen, and R³ or R⁴ amino, with the other onelower alkoxy.

[0165] 30. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen, and R³ or R⁴ lower mono or dialkylamino,with the other one lower alkoxy.

[0166] 31. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R⁴ lower mono or dialkylamino, with R³hydroxy.

[0167] Scheme 12—Route to Preferred Group 28

[0168] 5-Bromo-2,6-difluoronicotinic acid is prepared from2,6-difluoropyridine by successive lithiations using LDA. The 5-positionis alkylated via a Stille coupling, and the pyrimidone ring is cyclizedon in two steps. The 4-substituent is introduced in the usual fashionand the 7-fluoro group is displaced with thiomethoxide. This thioetherin turn is displaced by a Grignard agent in the presence of a nickelsalt catalyst. Again use of appropriate organometallic reagents in theStille and Grignard couplings could lead to alkenyl, alkynyl and arylsubstituents at R³ and R⁴.

[0169] 28. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen and R³ and R⁴ lower alkyl.

[0170] Scheme 13—Route to Preferred Groups 29 and 30, R⁴═RO

[0171] Nitration of the commercially available dichloronicotinic acid isfollowed by a selective displacement of the more reactive Cl under mildconditions, followed by a more forcing displacement of the other Cl, inthe appropriate order. The resulting 6-alkoxy-2-amino-5-nitronicotinicacid is cyclized to the pyrimidone, and the 4-carbonyl is converted to achloride and displaced in the usual fashion with an appropriate amine togive the 4-amino-7-alkoxy-6-nitropyrido[2,3-d]pyrimidine. Reduction ofthe nitro group, followed by any desired alkylation or acylation givesthe desired compounds.

[0172] Scheme 14—Route to Preferred Group 32

[0173] Compounds of group 32 are specialized cases of preferred groups27, 29, 30 and 31, where R³ and R⁴ are cyclized together. They can bemade using the same routes as those described for these preferred groupswith minor modifications. For example, vicinally substituted alkoxyamino compounds can be dealkylated, and the corresponding vicinal aminoalcohols can be bisalkylated with an appropriate dihaloalkane.Piperazines can be made by the route shown in Scheme 13, provided that asuitable amine nucleophile is used to displace the 6-chloro substituentinstead of an alkoxide.

[0174] 32. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, A,B & D carbon, with E nitrogen, and R³ and R⁴ taken together aredioxymethylene, dioxyethylene, 2,3-fused piperazine, 2,3-fusedmorpholine or 2,3-fused thiomorpholine.

[0175] Scheme 15—Route to Preferred Groups 33-36

[0176] Reaction of a suitable S-alkylisothiouronium salt withmethoxymethylidine malononitrile yields a fully functionalized pyrimideprecursor. The initially formed pyrimidine can have the SEt displaced byR⁴ either before or after the nitrile hydrolysis, if displacement oroxidation prove problematic later. Displacement of the SEt group canalso be achieved without an oxidation to activate the sulfur.Cyclization of the second pyrimidine ring is followed by activation ofthe 4-carbonyl by thiation and alkylation. Even if the 7-thio group hasnot been displaced at this point, introduction of the 4-amino sidechainoccurs preferentially.

[0177] 33. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, A & D carbon, with B and Enitrogen and R⁴ lower alkoxy.

[0178] 34. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, A & D carbon, with B and Enitrogen and R⁴ lower mono or dialkylamino.

[0179] 35. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, A & D carbon, with B and Enitrogen and R⁴ amino.

[0180] 36. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, A & D carbon, with B and Enitrogen and R⁴ hydrazino.

[0181] Scheme 16—Route to Preferred Groups 37-40

[0182] The pterine nucleus is made by well-established procedure. Forgroup 37, the pterindione intermediate can be O-alkylated, and for it,and the other groups, the pterindione can be converted to thetrichloropterin, and selective displacements can be carried out on thehalogens in an order appropriate to give the desired compound.

[0183] 37. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, B & D carbon, with A and Enitrogen and R³ and R⁴ lower alkoxy.

[0184] 38. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, B & D carbon, with A and Enitrogen and R³ and R⁴ lower mono or dialkylamino.

[0185] 39. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, B & D carbon, with A and Enitrogen and R³ or R⁴ lower alkoxy, with the other lower mono ordialkylamino.

[0186] 40. Another preferred form of the invention has X═NH, n=0, thearomatic ring phenyl optionally substituted, B & D carbon, with A and Enitrogen and R³ and R⁴ taken together are ethylenedioxy, 2,3-fusedpiperazine, 2,3-fused morpholine or 2,3-fused thiomorpholine.

[0187] Scheme 17—Route to Preferred Groups 41 [3,2-d] ring fusion

[0188] 3,H-Thieno[3,2-d]pyrimid-4-one can be made by standard chemistryfrom commercially available ethyl 3-aminothiophene carboxylate andformamide. Conversion of the carbonyl to chloride by standard techniquesfollowed by displacement with an appropriate amine gives the desiredthieno[3,2-d]pyrimidines. If R⁴ is not H, an appropriate electrophile,for example nitro for amine based or diazotization derived substituents,or Br for Stille coupled final products, can be introduced either at thestage shown or an earlier stage, and then be converted to R⁴, byreduction and amination for example or by Stille coupling, or othermethods known to those skilled in the art. [This technique follows alsofor all of the following preferred categories which have the possibilityof substitution on R³ or R⁴, as they are all contain electron rich fivemembered-rings which can be readily manipulated by electrophilicaromatic substitution.] (DMSO is dimethyl sulfoxide).

[0189] 41. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a sulfur atom, with D & E carbon, or A& B are carbon with D and E taken together as a sulfur atom, with R⁴ orR³ H, lower alkyl, lower alkoxy, amino, or lower mono or dialkylamino.

[0190] Scheme 18—Route to Preferred Groups 41 [2,3-d] ring fusion

[0191] Thieno[2,3-d]pyrimid-4-one is built up by the Gewald synthesisfrom 2,5-dihydroxy-1,4-dithiane and ethyl cyanoacetate, followed byformamide cyclization. Conversion of the carbonyl to chloride bystandard techniques followed by displacement with an appropriate aminegives the desired thieno[2,3-d]pyrimidines.

[0192] Scheme 19—Route to Preferred Groups 42 [3,2-d] ring fusion

[0193] The [3,2-d] ring fusion compounds are obtained from3-bromofurfural as shown above in Scheme A. Displacement of the bromideby azide, followed by oxidation of the aldehyde sets up the basicaminofuroic acid needed to fuse on the pyrimidine ring. The annulationshown can be used, or by manipulating which acid derivative is actuallyused, one could use a variety of other ring annulations, and subsequentactivations of the 4-position if required.

[0194] 42. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are an oxygen atom, with D & E carbon, orA & B are carbon with D and E taken together as an oxygen atom, with R⁴or R³ H, lower alkyl, lower alkoxy, amino, or lower mono ordialkylamino.

[0195] Scheme 20—Route to Preferred Groups 42 [2,3-d] ring fusion

[0196] Reaction of 6-chloro-4-methylthiopyrimidine with LDA followed byDMF gives the corresponding 5-aldehyde, which is treated with the sodiumsalt of an appropriate glycollate ester, displacing chlorine, and insitu forming the furan ring by intramolecular aldol condensation.Cleavage of the ester and decarboxylation of the unwanted 7-acidfunctionality may be done in a single reaction with a good nucleophilein a dipolar aprotic solvent at high temperature, or in separatesaponification and Cu/guinoline decarboxylation steps. Displacement ofthe 4-methylthio group by an appropriate amine gives the desiredfurano[2,3-d]pyrimidines.

[0197] Scheme 21—Route to Preferred Groups 43 [2,3-d] ring fusion

[0198] To make the pyrrolo[2,3-d]pyrimidine a pyrimidine ring iscyclized onto the cyano amino-pyrrole using known techniques as shown inscheme B above. Activation and displacement of the thiol by the sidechain can be preceded or followed by the optional electrophilicsubstitution of the pyrrole ring.

[0199] 43. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a nitrogen atom, with D & E carbon, orA & B are carbon with D and E taken together as a nitrogen atom, with R⁴or R³ H, lower alkyl, lower alkoxy, amino, or lower mono ordialkylamino.

[0200] Scheme 22—Route to Preferred Groups 43 [3,2-d] ring fusion

[0201] The preparation of the pyrrolo[3,2-d]pyrimidine exploits theknown condensation of orthoformate with the acidified 4-methyl group of6-pyrimidones to form the pyrrolopyrimidine as shown above. The sidechain can be put on by standard techniques such as in Scheme 1, and theR⁴ substituent can be introduced by standard electrophilic chemistry asdescribed above.

[0202] Scheme 23—Route to Preferred Groups 44 [5,4-d] ring fusion

[0203] Condensation of dithioformic acid with 2-aminomalononitrile inthe presence of a dehydrating agent such as PPA gives5-amino-4-cyanothiazole. Reaction of this with orthoformate, followed bytreatment with MeSNa gives a thiazolo[5,4-d]pyrimidine derivative, whichon treatment with an appropriate amine give the desired compounds.

[0204] 44. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a sulfur atom with D carbon and Enitrogen, or D and E taken together are a sulfur atom, and A is nitrogenand B is carbon, with R^(¾) H, lower alkyl, lower alkoxy, amino, orlower mono or dialkylamino.

[0205] Scheme 24—Route to Preferred Groups 44 [4,5-d] ring fusion

[0206] Reaction of N-cyanobismethylthiomethyl-eneimine with ethylthioglycollate gives ethyl 2-S methylthio-4-aminothiazole-5-carboxamide.Cyclization with formamide or equivalent, followed by desulfurization ofthe methylthio gives a thiazolopyrimidone, which can be activated byVilsmeier reagent, and the chloride displaced by the desired amine togive the desired thiazolo[4,5-d]pyrimidine derivatives as shown above.

[0207] Scheme 25—Route to Preferred Groups 45 [5,4-d] ring fusion

[0208] The known 5-amino-4-cyanooxazole is treated with ethylorthoformate/acetic anhydride, and is then reacted with MeSNa to give4-methylthiooxazolo[5,4-d]pyrimidine, which on displacement with theappropriate amine gives the desired oxazolo[5,4-d]pyrimidines as shownabove.

[0209] 45. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are an oxygen atom with D carbon and Enitrogen, or D and E taken together are an oxygen atom, and A isnitrogen and B is carbon, with R^(¾) H, lower alkyl, lower alkoxy,amino, or lower mono or dialkylamino.

[0210] Scheme 26—Route to Preferred Groups 45 [4,5-d] ring fusion

[0211] Diazotization of the known 5-amino-4,6-dichloropyrimidine,followed-by dilute sulfuric acid treatment give the corresponding5-hydroxy compound. One of the chlorines is displaced with ammonia, andthe oxazole ring is annulated with formic acid or an appropriateequivalent. Displacement of the other chlorine with an appropriate aminegives the desired oxazolo[4,5-d]pyrimidines as shown above

[0212] Scheme 27—Route to Preferred Groups 46

[0213] These compounds can be made by straightforward displacement ofhalogen on appropriate 6-chloropurines, by means well documented in theart. R³ substituents can be introduced via facile electrophilicsubstitutions at the activated 8-position of the purine nucleus,followed by the types of transformation discussed in previous examples.

[0214] 46. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, Aand B taken together are a nitrogen atom, and D is carbon and E isnitrogen, with R^({fraction (3/6)}) H, or lower alkyl, and R⁴ H, loweralkyl, lower alkoxy, amino, or lower mono or dialkylamino.

[0215] Scheme 28—Route to Preferred Groups 47 [5,4-d] ring fusion

[0216] Reaction of 6-chloro-4-methylthiopyrimidine with LDA followed byDMF gives the corresponding 5-aldehyde, which is treated withhydroxylamine under mild acidic conditions, and then basic conditions tocomplete the ring annulation giving4-methylthioisoxazolo[5,4-d]pyrimidine, which on displacement with anappropriate amine gives the desired isoxazolo[5,4-d]pyrimidinederivatives as shown above.

[0217] 47. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are an oxygen atom with D nitrogen and Ecarbon, or A and B taken together are a carbon atom with D nitrogen andE oxygen, with R^({fraction (3/6)}) H, lower alkyl, lower alkoxy, amino,or lower mono or dialkylamino.

[0218] Scheme 29—Route to Preferred Groups 47 [4,5-d] ring fusion

[0219] Reaction of 4,6-dichloro-5-nitropyrimidine with CuCN/NMP givesthe 4-nitrile. Reduction of the nitro group to the corresponding amineis followed by diazotization and treatment with dilute sulfuric acid togive the corresponding 5-hydroxy compound. Reaction of this withMe₃Al/NH₄Cl gives the amidine which is oxidatively cyclized to7-amino-4-chloroisoxazolo[4,5-d]pyrimidine. Removal of the aminofunctionality by diazotization/hypophosphorus acid is followed bydisplacement of the 4-chlorine with an appropriate amine to give thedesired isoxazolo[4,5-d]pyrimidine derivatives as shown above.

[0220] Scheme 30—Route to Preferred Groups 48 [5,4-d] ring fusion

[0221] Reaction of 6-chloro-4-methylthiopyrimidine with LDA followed byDMF gives the corresponding 5-aldehyde, which is treated sequentiallywith NaSH, NBS and ammonia to complete the ring annulation giving4-methylthioisothiazolo[5,4-d]pyrimidine, which on displacement with anappropriate amine gives the desired isothiazolo[5,4-d]pyrimidinederivatives as shown above.

[0222] 48. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a sulfur atom with D nitrogen and Ecarbon, or A and B taken together are a carbon atom with D nitrogen andE sulfur, with R^({fraction (3/6)}) H, lower alkyl, lower alkoxy, amino,or lower mono or dialkylamino.

[0223] Scheme 31—Route to Preferred Groups 48 [4,5-d] ring fusion

[0224] Reaction of 4,6-dichloro-5-nitropyrimidine with CuCN/NMP givesthe 4-nitrile. Reduction of the nitro group to the amine is followed bydiazotization/thiation to give the corresponding 5-mercapto compound.Reaction of this with Me₃Al/NH₄Cl gives the amidine which is oxidativelycyclized with NBS to 7-amino-4-chloroisothiazolo[4,5-d]pyrimidine.Removal of the amino functionality by diazotization/hypophosphorus acidis followed by displacement of the 4-chlorine with an appropriate amineto give the desired isothiazolo4,3-d]pyrimidine derivatives as shownabove.

[0225] Scheme 32—Route to Preferred Groups 49-[3,4-d] ring fusion

[0226] Reaction of-6-chloro-4-methylthiopyrimidine with LDA followed byDMF gives the corresponding 5-aldehyde, which is treated with hydrazineto do the ring annulation giving 4-methylthiopyrazolo[3,4-d]pyrimidine,which on displacement with an appropriate amine gives the desiredpyrazolo[3,4-d]pyrimidine derivatives as shown above.

[0227] 49. Another preferred form of the invention has X═NH, n=0 or 1,in which case R¹═H, the aromatic ring phenyl optionally substituted, andeither A and B taken together are a nitrogen atom with D nitrogen and Ecarbon, or A and B taken together are a carbon atom with D and Enitrogen atoms, with R^({fraction (3/6)}) H or lower alkyl if onnitrogen, or H, lower alkyl, lower alkoxy, amino, or lower mono ordialkylamino if on carbon.

[0228] Scheme 33—Route to Preferred Groups 49 [4,3-d] ring fusion

[0229] Nitration of pyrazole-3-carboxylic acid followed by reductiongives 4-aminopyrazole-3-carboxylic acid. This is cyclized topyrazolo[4,3-d]pyrimid-4-one with formamidine HCl, and replacement ofthe carbonyl with halide by standard procedures, followed bydisplacement of the chloride by an appropriate amine yields the desiredpyrazolo[4,3-d]pyrimidine, as shown above.

[0230] Most Preferred Forms of the Invention

[0231] 1. A most preferred form of the invention is one where X═NH, x=0,the aromatic ring is 3-bromophenyl, B, D & E are carbon, A is nitrogen,and R⁴ is amino.

[0232] 2. A most preferred form of the invention is one where X═NH, x=0,the aromatic ring is 3-bromophenyl, B, D & E are carbon, A is nitrogen,and R⁴ is methylamino.

[0233] 3. A most preferred form of the invention is one where X═NH, x=0,the aromatic ring is 3-bromophenyl, B, D & E are carbon, A is nitrogen,and R⁴ is dimethylamino.

[0234] 4. A most preferred form of the invention is one where X═NH, x=0,the aromatic ring is 3-nitrophenyl, A, D & E are carbon, B is nitrogen,and R⁴ is amino.

[0235] 5. A most preferred form of the invention is one where X═NH, x=0,the aromatic ring is 3-bromophenyl, A, D & E are carbon, B is nitrogen,and R⁴ is amino.

[0236] 6. A most preferred form of the invention is one where X═NH, x=0,the aromatic ring is 4-bromophenyl, A, D & E are carbon, B is nitrogen,and R⁴ is amino.

[0237] 7. A most preferred form of the invention is one where X═NH, x=0,the aromatic ring is 3-trifluoromethylphenyl, A, D & E are carbon, B isnitrogen, and R⁴ is amino.

[0238] 8. A most preferred form of the invention is one where X═NH, x=0,the aromatic ring is 3-bromophenyl, A, D & E are carbon, B is nitrogen,and R⁴ is acetylamino.

[0239] 9. A most preferred form of the invention is one where X═NH, x=1,R¹═H, the aromatic ring is phenyl, A, D & E are carbon, B is nitrogen.

[0240] 10. A most preferred form of the invention is one where X═NH,x=1, R¹═H, the aromatic ring is phenyl, A, D & E are carbon, B isnitrogen, and R⁴ is acetylamino.

[0241] 11. A most Preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, B & E are carbon, D isnitrogen, R³=Cl.

[0242] 12. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, D & E are carbon, D isnitrogen, and R³ is methoxy.

[0243] 13. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, D & E are carbon, D isnitrogen, and R³ is methylamino.

[0244] 14. A most Preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, D & E are carbon, D isnitrogen, and R³ is dimethylamino.

[0245] 15. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, D & E are carbon, and A and Btaken together are S.

[0246] 16. A most preferred form of the invention is one where X═NH,x=1, R¹═H, the aromatic ring is phenyl, D & E are carbon, and A and Btaken together are S.

[0247] 17. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A & B are carbon, and D and Etaken together are S.

[0248] 18. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, B is carbon, and A, and D and Etaken together, are nitrogen.

[0249] 19. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, B & E are carbon, D isnitrogen, and R³ is N-piperinyl.

[0250] 20. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, D & E are carbon, B isnitrogen, and R⁴ is fluoro.

[0251] 21. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-hydroxyphenyl, A, D & E are carbon, B isnitrogen, and R⁴ is amino.

[0252] 22. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, D-& E are carbon, B isnitrogen, and R⁴ is methylamino.

[0253] 23. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, D & E are carbon, B isnitrogen, and R⁴ is dimethylamino.

[0254] 24. A most preferred form of the invention is one where X═NMe,x=0, the aromatic ring is 3-bromophenyl, A, D & E are carbon, B isnitrogen, and R⁴ is methylamino.

[0255] 25. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, D & E are carbon, B isnitrogen, and R⁴ is methoxy.

[0256] 26. A most preferred form of the invention is one where X═NH,x=0, the aromatic ring is 3-bromophenyl, A, B & D are carbon, E isnitrogen, and R⁴ is fluoro.

[0257] Biology

[0258] These compounds are potent and selective inhibitors of the humanEGF receptor tyrosine kinase, and other members of the EGF receptorfamily, including the ERB-B2, ERB-B3 and ERB-B4 receptor kinases, andare useful for the treatment of proliferative diseases in mammals. Theseinhibitors prevent mitogenesis in cells where mitogenesis is driven byone or more of this family of receptor kinases. This can include normalcells, where it is desired to prevent mitogenesis, as exemplified by thecells transformed by overexpression or mutation of this kinase family asexemplified by poor prognosis breast cancer where overexpression ofEGFR, ERB-B2 and ERB-B3 or mutation of ERB-B2 to the oncoprotein NEU isa major factor in cellular transformation. As the Preferred compoundsare not highly cytotoxic and do not show potent growth inhibitoryproperties, because of their high specificity toward inhibition of theEGFR kinase family, they should have a much cleaner toxicity profilethan most anti-cancer and anti-proliferative drugs. Their very differentmode of action to current anti-cancer drugs should allow for their usein multiple drug therapies, where synergism with available agents isanticipated.

[0259] Compounds of the invention have been shown to be very potent,reversible inhibitors of the EGF receptor tyrosine kinase, by bindingwith high affinity at the adenosine triphosphate (ATP) binding site ofthe kinase. These compounds exhibit potent IC₅₀s, varying from 10micromolar to 5 picomolar, for the tyrosine kinase activity of theenzyme, based on an assay examining phosphorylation of a peptide derivedfrom the phosphorylation site of the protein PLCgammal, a known EGFRphosphorylation substrate. This data is shown in Table 1.

Biological Data

[0260] Materials and Methods

[0261] Purification of Epidermal Growth Factor Receptor TyrosineKinase—Human EGF receptor tyrosine kinase was isolated from A431 humanepidermoid carcinoma cells which overexpress EGF receptor by thefollowing methods. Cells were grown in roller bottles in 50% Delbuco'sModified Eagle and 50% HAM F-12 nutrient media (Gibco) containing 10%fetal calf serum. Approximately 10⁹ cells were lysed in two volumes ofbuffer containing 20 mM2-(4N-[2-hydroxyethyl]piperazin-1-yl)ethanesulfonic acid (hepes), pH7.4, 5 mM ethylene glycol bis(2-aminoethyl ether) N,N,N′,N′-tetraaceticacid, 1% Triton X-100, 10% glycerol, 0.1 mM sodium orthovanadate, 5 mMsodium fluoride, 4 mM pyrophosphate, 4 mM benzamide, 1 mMdithiothreitol, 80 μg/mL aprotinin, 40 μg/mL leupeptin and 1 mMphenylmethylsulfonyl fluoride. After centrifugation at 25,000 x g for 10minutes, the supernatant was equilibrated for 2 h at 4° C. with 10 mL ofwheat germ agglutinin sepharose that was previously equilibrated with 50mM Hepes, lo glycerol, 0.1% Triton X-100 and 150 mM NaCl, pH 7.5,(equilibration buffer). Contaminating proteins were washed from theresin with 1 M NaCl in equilibration buffer, and the enzyme was elutedwith 0.5 M N-acetyl-1-D-glucosamine in equilibration buffer, followed by1 mM urea. The enzyme was eluted with 0.1 mg/ml EGF. The receptorappeared to be homogeneous as assessed by Coomassie blue stainedpolyacrylamide electrophoretic gels.

[0262] Determination of 1C₅₀ values—enzyme assays for IC₅₀determinations were performed in a total volume of 0.1 mL, containing 25mM Hepes, pH 7.4, 5 mM MgCl₂, 2 mM MnCl₂, 50 μM sodium vanadate, 5-10 ngof EGF receptor tyrosine kinase, 200 μM of a substrate peptide,(Ac-Lys-His-Lys-Lys-Leu-Ala-Glu-Gly-Ser-Ala-Tyr⁴⁷²-Glu-Glu-Val—NH₂,derived from the amino acid (Tyr⁴⁷² has been shown to be one of fourtyrosines in PLC (phospholipaseC)-gamma 1 that are phosphorylated by theEGF receptor tyrosine kinase [Wahl, M. I.; Nishibe, S.; Kim, J. W.; Kim,H.; Rhee, S. G.; Carpenter, G., J. Biol. Chem., (1990), 265,3944-3948.], and peptides derived from the enzyme sequence surroundingthis site are excellent substrates for the enzyme.), 10 μM ATPcontaining 1 μCi of [³²P]ATP and incubated for ten minutes at roomtemperature. The reaction was terminated by the addition of 2 mL of 75mM phosphoric acid and passed through a 2.5 cm phosphocellulose filterdisc to bind the peptide. The filter was washed five times with 75 mMphosphoric acid and placed in a vial along with 5 mL of scintillationfluid (Ready gel Beckman). TABLE 1 EGF Receptor Tyrosine KinaseInhibition Example # IC₅₀  1 8 μM  2 3.6 μM  3 1.1 μM  4 225 nM  5 1.9μM  6 7.6 nM  7 3.1 nM  8 9.6 nM  9 405 nM 10 6.1 μM 11 194 nM 12 13 nM13 250 nM 14 70 nM 15 134 nM 16 3.7 μM 17 1.55 μM 18 173 nM 19 1.8 μM 204.9 μM 21 1.25 μM 22 39 nM 23 840 nM 24 123 nM 25 377 nM 26 241 nM 27 10nM 28 94 nM 29 262 nM 30 10 μM 31 15 nM 32 4.7 μM 33 130 pM 34 91 pM 353.1 nM 36 29 nM 37 39 nM 38 71 nM 39 590 nM 40 578 nM 41 220 nM 42 226nM 43 10 μM 44 10 μM 45 2.87 μM 46 1.42 μM 47 1.67 μM 48 1.0 μM 49 2.5μM 50 10 μM 51 1.95 μM 52 8 μM 53 1.8 μM 54 100 nM 55 400 nM 56 110 nM57 124 nM 58 40 nM 59 2.6 nM 60 8 pM 61 6 pM 62 6.1 μM 63 6.1 μM 64 11nM 65 5.1 μM 66 190 nM 67 6.1 μM 68 263 nM 69 7.0 μM 70 473 nM 71 11 nM72 35 nM 73 36 nM 74 11.5 μM 75 55 nM 76 10 μM 77 3 nM 78 670 nM 79 6.7nM

[0263] Swiss 3T3 mouse fibroblasts, A431 human epidermoid carcinomacells, and MCF-7 (Michigan Cancer Foundation human mammary carcinomacells), SK-BR-3 (human mammary carcinoma cells), MDA-M3-231 andMDA-MB-468 (human mammary carcinoma cells) breast carcinomas wereobtained from the American Type Culture Collection, Rockville, Md. andmaintained as monolayers in dMEM (Dulbecco's modified eagle medium)/F12,50:50 (Gibco/BRL) containing 10% fetal bovine serum. To obtainconditioned medium, MDA-MB-231 cells were grown to confluency in an 850cm² roller bottle and the medium-replaced with 50 ml of serum-freemedium. After 3 days the conditioned medium was removed, frozen down inaliquots and used as a heregulin source to stimulate erb-2, 3, 4.

[0264] Antibodies

[0265] Monoclonal antibodies raised to the PDGF (platelet-desired growthfactor) receptor or phosphotyrosine were from Upstate Biotechnology,Inc., Lake Placid, N.Y. Anti-pp39^(jun) (antibody to the transcriptionfactor c-jun, which is a 39 kDalton phosphoprotein) and anti-EGFreceptor antibodies were from Oncogene Science, Uniondale, N.Y.

[0266] Immunoprecipitation and Western Blot

[0267] Cells were grown to 100% confluency in 100 mm Petrie dishes(Corning). After the cells were treated for 5 minutes with either EGF(epidermal growth factor), PDGF, or bFGF (basic fibroblast growthfactor) (20 ng/ml) or 1 ml of conditioned media from MDA-MB-231 cells,the media was removed and the monolayer scraped into 1 ml of ice coldlysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% tritonX-100, 1 mM EDTA, 1 mM EGTA, 10 mM sodium pyrophosphate, 30 mMp-nitrophenyl phosphate, 1 mM orthovanadate, 50 mM sodium fluoride, 1 mMphenylmethylsulfonylfluoride, 10 μg/ml of aprotinin, and 10 μg/ml ofleupeptin). The lysate was transferred to a microfuge tube (smallcentrifuge that holds 1-2 ml plastic centrifuge tubes), allowed to siton-ice 15 minutes and centrifuged 5 minutes at 10,000 x g. Thesupernatant was transferred to a clean microfuge tube and 5 μg ofantibody was added to designated samples. The tubes were rotated for 2hours at 4° C. after which 25 μl of protein A sepharose was added andthen rotation continued for at least 2 more hours. The protein Aseparose was washed 5 times with 50 mM Hepes, pH 7.5, 150 mM NaCl, 10%glycerol and 0.02% sodium azide. The precipitates were resuspended with30 μl of Laemlli buffer (Laemmli, NATURE, Vol. 727, pp. 680-685, 1970),heated to 100° C. for 5 minutes and centrifuged to obtain thesupernatant. Whole cell extracts were made by scraping cells grown inthe wells of 6 well plates into 0.2 ml of boiling Laemmli buffer. Theextract were transferred to a microfuge tube and heated to 100° C. for 5minutes. The entire supernatant from the immunoprecipitation or 35 μl ofthe whole cell extract was loaded onto a polyacrylamide gel (4-20%) andelectrophoresis carried out by the method of Laemlli (Laemmli, 1970).Proteins in the gel were electrophoretically transferred tonitrocellulose and the membrane was washed once in 10 mM Tris buffer, pH7.2, 150 mM NaCl, 0.01% Azide (TNA) and blocked overnight in TNAcontaining 5% bovine serum albumin and 1% ovalbumin (blocking buffer).The membrane was blotted for 2 hours with the primary antibody (1 μg/mlin blocking buffer) and then washed 2 times sequentially in TNA, TNAcontaining 0.05% Tween-20 and 0.05% Nonidet P-40 (commercially availabledetergent) and TNA. The membranes were then incubated for 2 hours inblocking buffer containing 0.1 μCi/ml of [¹²⁵I] protein A and thenwashed again as above. After the blots were dry they were loaded into afilm cassette and exposed to X-AR X-ray film for 1-7 days. Protein A isa bacterial protein that specifically bonds certain IgG subtypes and isuseful in binding to and isolating antibody-antigen complexes.

[0268] Northern Blots

[0269] Total cellular RNA was isolated from untreated control or treatedSwiss 3T3 cells using RNAzol-B (trademark of Tel Test Inc. for a kitused to isolate RNA from tissues) and adhered to the protocol describedby the manufacturer. Forty to fifty μg of RNA was loaded onto a 1%agarose gel and electrophoresis carried out for 3-4 hours at 65 volts.The RNA in the gel was transferred by capillary action to a nylonmembrane (Hybond-N, Amersham). The 40 mer c-jun probe was end labeledwith [³²P]ATP using T4 nucleotide kinase (Promega) and purified on a G25sephadex column according to the procedure recommended by the supplier,Oncogene Science. Hybridization was performed overnight at 65° C. (c-junis an immediate early transcription factor; it is one of the componentsof AP-1 while FOS is the second component of AP-1.

[0270] Growth Factor-Mediated Mitogenesis

[0271] Swiss 3T3 fibroblasts were grown to 90 -100% confluency in 24-well plates (1.7×1.6 cm, flat bottom) and growth arrested in serum-freemedia for 18 hours. Drug was added to specified wells 2 hours prior togrowth factors and then the cells were exposed to either 20 ng/ml EGF,PDGF or bFGF or 10% serum for 24 hours. Two μCi of [methyl-³H]thymidinewas added to each well and incubated for 2 hours at 37° C. The cellswere trypsinized and injected into 2 ml of ice cold 15% trichloroaceticacid (TCA). The resulting precipitate was collected on glassfiberfilters, washed five times with 2-ml aliquots of ice-cold 15% TCA, driedand placed in scintillation vials along with 10 ml Ready gel (Beckman,Irvine, Calif.). Radioactivity was determined in a Beckman LS 6800scintillation counter.

[0272] Growth Inhibition Assay

[0273] Cells (2×10⁴) were seeded in 24-well plates (1.7×1.6 cm, flatbottom) in two mls of medium with or without various concentrations ofdrug. Plates were incubated for 3 days at 37° in a humidified atmospherecontaining 5 CO₂ in air. Cell growth was determined by cell count with aCoulter Model AM electronic cell counter (Coulter Electronics, Inc.,Hialeah, Fla.).

INHIBITION OF EGF-INDUCED AUTOPHOSPEORYLATION IN A431 EPIDERMOIDCARCINOMA CELLS AND CONDITIONED MEDIA-INDUCED AUTOPHOSPHORYLATION INSK-BR-3 BREAST TUMOR CELLS BY COMPOUNDS OF THE CURRENT INVENTION

[0274] Example # EGFR IC₅₀ nM A431 IC₅₀ nM SKBR-3 IC₅₀ nM 4 225 <1000<10,000 6 7.6 53 2660 7 3.1 20 100 8 9.6 32 71 22  39 252 −1500 27  10110 −800 59  2.6 12 <10 60  0.008 13 <10 61  0.006 21 39 70  11 124 <1074  55 <1000 <1000

ANTIPROLIFERATIVE PROPERTIES OF TYROSINE KINASE INHIBITORS IC₅₀ (nM)

[0275] Ex 60 Ex 61 B104-1-1 2100  1000 SK-B-3  600  900 MDA-468 300012000

[0276] B104-1-1—NIH-3T3 fibroblasts transfected by the neu oncogene,Stem et al., Science, 234, pp. 321-324 (1987)

[0277] SK-BR-3—Human breast carcinoma overexpressing erbB-2 and erbB-3

[0278] MDA-468—Human breast carcinoma overexpressing the EGF receptor

[0279] The above gels, developed as detailed in the experimentalsection, demonstrate the efficacy of compounds of the current inventionat blocking certain EGF-stimulated mitogenic signalling events in wholecells. The numbers to the left of the gels indicated the positions ofmolecular weight standards in kiloDaltons. The lane labelled controlshows the degree of expression of the growth-related signal in theabsence of EGF stimulation, whereas the lane labelled EGF (or PDGF orb-FGF) shows the magnitude of the growth factor-stimulated signal. Theother lanes show the effect of the stated quantities of the named drugon the growth factor-stimulated activity being measured, demonstratingthat the compounds of the present invention have potent effects in wholecells, consistent with their ability to inhibit the tyrosine kinaseactivity of the EGF receptor.

[0280] Gel of Example 40 (FIG. 7) detects mRNA-for c-jun byhybridization with a specific radiolabelled RNA probe for c-jun. The geldemonstrates that the growth factors EFG, PDGF and b-FGF stimulate c-junproduction in Swiss 3T3 cells, and that compound 40 blocks thisproduction for EGF-stimulated cells, but not for PDGF or b-FGFstimulated cells.

[0281] Effect of Example 40 on Growth Factor Mediated Expression ofp39^(c-jun)

[0282] This gel shows the amount of c-jun induced in Swiss 3T3 cells bythe growth factor EGF, PDGF and b-FGF, quantitating with ananti-c-jun-specific monoclonal antibody. It demonstrates the ability ofExample 40 to block c-jun expression in Swiss 3T3 when stimulated byEGF, but not when stimulated by PDGF or b-FGF.

[0283] It is to be appreciated that the compounds described herein canbe used in combination with other components to enhance their activity.Such additional components are anti-neoplastic materials as,doxorubicin, taxol, cis platin, and the like.

[0284] It has been found that the compounds described herein may inhibitboth the erb-B2 and erb-B4 receptors and therefore have significantlyincreased clinical activity advantageously in combination with theaforementioned anti-neoplastic agents.

[0285] See also the results shown in FIGS. 1-17.

[0286] Some preferred structures are as follows:

Chemical Experimental

[0287] Listed below are preferred embodiments wherein all temperaturesare in degrees Centigrade and all parts are parts by weight unlessotherwise indicated.

EXAMPLE 1

[0288] 4-Anilinopyrido[3,2-d]pyrimidine mesylate

[0289] 3H-Pyrido[3,2-d]pyrimidin-4-one. A solution of6-chloro-3-nitropicolinamide (2.00 g, 9.91 mmol) in EtOAc/MeOH (1:1, 100mL) is hydrogenated over 5% Pd-C (0.40 g) at 60 psi for 6 days, withadditions of fresh catalyst after 2 and 4 days. After removal of thecatalyst by filtration the solution is concentrated to dryness, to give3-aminopicolinamide as an orange oil, which is used directly in the nextstep. The crude product is stirred under reflux with triethylorthoformate (50 mL) for 42 h, during which time a tan precipitateforms. After cooling, the solid is filtered off, washed well withpetroleum ether, and dried under vacuum to give3H-pyrido[3,2-d]pyrimidin-4-one (1.27 g, 87%), mp 343-345° C. [Price, C.C. and Curtin, D. Y. J. Amer. Chem. Soc. 68, 914, 1946 report mp346-347° C.].

[0290] 4-Chloropyrido[3,2-d]pyrimidine. A suspension of the abovepyrimidinone (1.00 g, 6.80 mmol) in POCl₃ (30 mL) is heated under refluxfor 4 h, and then concentrated to dryness under reduced pressure. Theresidue is partitioned between CH₂Cl₂ and saturated NaHCO₃ solution, andthe organic layer worked up to give 4-chloropyrido[3,2-d]pyrimidine(0.97 g, 86%) as a tan solid, mp 335° C. (dec), which is used withoutfurther characterisation.

[0291] 4-Anilinopyrido[3,2-d]pyrimidine mesylate.

[0292] A solution of 4-chloropyrido[3,2-d]pyrimidine (84 mg, 0.5 mmol),aniline (56 mg, 0.6 mmol) and triethylamine (62 mg, 0.6 mmol) in EtOH (2mL) are refluxed under N₂ with stirring for 2 h. The crude reactionmixture is purified on a preparative tlc plate (silica), eluting oncewith 3% MeOH in CHCl₃. The major band is extracted, and evaporated todryness under reduced pressure, and the residual solid is dissolved inacetone, (5 mL), filtered, and methanesulfonic acid (32 μL, 0.5 mmol) isadded slowly with swirling. The precipitate is collected by suctionfiltration, rinsed with acetone and dried in a vacuum oven to give4-anilinopyrido[3,2-d]pyrimidine mesylate (91 mg, 57%) as dull yellowneedles. ¹H NMR (DMSO) δ 11.75 (1H, slbrs), 9.11 (1H, dd, J=1.5, 4.3Hz), 8.97 (1H, s), 8.32 (1H, dd, J=1.5, 8.4 Hz), 8.12 (1H, dd, J=4.3,8.5 Hz), 7.88 (2H, d, J=8.2 Hz), 7.49 (2H, t, J=8.0 Hz), 7.32 (1H, t,J=7.0 Hz), 2.34 (3H, s)

EXAMPLE 2

[0293] 4-Benzylaminopyrido[3,2-d]pyrimidine

[0294] A solution of freshly prepared 4-chloropyrido[3,2-d]pyrimidine(0.10 g, 0.60 mmol) (prepared as described in the previous experimental)and benzylamine (0.13 mL, 1.20 mmol) in propan-2-ol (15 mL) containing atrace of conc. HCl is warmed at 50° C. for 30 min, and then concentratedto dryness. The residue is partitioned between water and EtOAc, and theorganic layer-worked up and chromatographed on silica gel. EtOAc elutesforeruns, while MeOH/EtOAc (1:9) elutes4-(benzylamino)pyrido[3,2-d]pyrimidine (0.11 g, 77%). ¹H NMR (CDCl₃) δ8.67 (1H, s), 6.50 (1H, dd, J=4.3, 1.5 Hz), 8.10 (1H, dd, J=8.5, 1.5Hz), 7.63 (1H, dd, J=8.8, 4.3 Hz), 7.55 (1H, brs) 7.41-7.29 (5H, m),4.86 (2H, d, J=5.9 Hz).

EXAMPLE 3

[0295] 4-(3-Bromoanilino)pyrido[3,2-d]pyrimidine

[0296] Reaction of 4-chloropyrido[3,2-d]pyrimidine (prepared asdescribed in a previous experimental) with 3-bromoaniline in propan-2-olcontaining a trace of conc. HCl at 50° C. for 30 min, followed bychromatography of the product on silica gel, gives4-(3-bromophenyl)aminopyrido[3,2-d]pyrimidine (87% yield). ¹H NMR(CDCl₃) δ 9.19 (1H, brs), 8.83 (1H, s), 8.80 (1H, dd, J=4.3, 1.5 Hz),8.29 (1H, brs), 8.19 (1H, dd, J=8.5, 1.5 Hz), 7.83 (1H, m), 7.76 (1H,dd, J=8.5, 4.3 Hz), 7.29-7.27 (2H, m).

EXAMPLE 4

[0297] 4-(3-Bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine

[0298] 2-cyano-6-fluoro-3-nitropyridine. A mixture of6-chloro-2-cyano-3-nitropyridine [Colbry, N. L.; Elslager, E. F.;Werbel, L. M.; J. Het. Chem., 1984, 21, 1521-1525](10.0 g, 0.054 mol)and KF (9.48 g, 0.163 mol) in MeCN (200 mL) is heated under reflux withstirring for 18 h, then poured into water and extracted with EtOAc. Theextract is washed with water and worked up, and the residue ischromatographed on silica gel, eluting with EtOAc/petroleum ether (3:7),to give after removal of the solvent under reduced pressure2-cyano-6-fluoro-3-nitropyridine (7.2 g, 79%). ¹H NMR (CDCl₃) δ 8.79(1H, dd, J=9.0, 6.0 Hz) 7.48 (1H, dd, J=9.0, 3.0 Hz).

[0299] 6-Fluoro-3-nitropyridine-2-carboxamide. A solution of2-cyano-6-fluoro-3-nitropyridine (1.40 g, 8.39 mmol) in 90% H₂SO₄ (30mL) is warmed at 70° C. for 90 min, then cooled, poured onto ice andbasified with conc. ammonia. Extraction with EtOAc and workup gives6-fluoro-3-nitropyridine-2-carboxamide (0.94 g, 61%). ¹H NMR (CDCl₃) δ8.70 (1H, dd, J=8.9, 6.5 Hz), 8.30, 8.03 (1H, 1H, brs), 7.62 (1H,. dd,J=8.9, 2.9 Hz).

[0300] 6-Fluoro-3H-pyrido[3,2-d]pyrimid-4-one. A solution of6-fluoro-3-nitropyridine-2-carboxamide (1.50 g, 8.10 mmol) in EtOAc (80mL) is hydrogenated over 5% Pd-C (0.30 g) at 60 psi for 2 h. Afterremoval of the catalyst by filtration, the solvent is removed underreduced pressure, to give a residue of crude3-amino-6-fluoropyridine-2-carboxamide which is used directly in thenext step. Triethyl orthoformate (60 mL) is added and the mixture isthen heated under reflux with vigorous stirring for 18 h. The cooledmixture is diluted with an equal volume of petroleum ether, and theresulting precipitate collected by filtration and is washed well withpetroleum ether to give 6-fluoro-3H-pyrido[3,2-d]pyrimid-4-one (1.26 g,84%). ¹H NMR (DMSO) δ 12.72 (1H, brs), 8.31 (1H, dd, J=8.6, 7.7 Hz),8.20 (1H, s), 7.66 (1H, dd, J=8.6, 3.0 Hz).

[0301] 4-(3-Bromoanilino)-6-fluoropyrido[3,2-d)pyrimidine. A suspensionof 6-fluoro-3H-pyrido[3,2-d]pyrimid-4-one (0.20 g, 1.21 mmol) in POCl₃(30 mL) is heated under reflux with stirring until homogeneous (2 h),and then for a further 1 h. Excess POCl₃ is removed under reducedpressure, and the residue is partitioned between CH₂Cl₂ and saturatedaqueous NaHCO₃. Workup of the organic portion gives crude4-chloro-6-fluoropyrido[3,2-d]pyrimidine (100%) as an unstable whitesolid which is used directly in the next step.

[0302] A solution of 4-chloro-6-fluoropyrido[3,2-d]pyrimidine (0.20 g,1.1 mmol) and 3-bromoaniline (0.12 mL, 2.18 mmol) in propan-2-ol (20 mL)containing conc. HCl (1 drop) is heated under reflux for 15 min, thencooled, poured into water and extracted with EtOAc. The extract isworked up, and the residue chromatographed on silica gel, eluting withEtOAc/petroleum ether (1:2) to give after removal of the solvent underreduced pressure 4-(3-bromoanilino)-6-fluoropyrido(3,2-d)pyrimidine(0.18 g, 52%). ¹H NMR (CDCl₃) δ 8.82 (1H, s), 8.65 (1H, brs), 8.31 (1H,t, J=7.4 Hz), 8.27 (1H, brs), 7.77 (1H, m) 7.41 (1H, dd, J=8.9, 2.2 Hz),7.29 (2H, brs).

EXAMPLE 5

[0303] 4-(3-Bromoanilino)-6-chloropyrido[3,2-d]pyrimidine

[0304] 6-chloro-3-nitropicolinamide. A solution of6-chloro-3-nitropicolinonitrile (1.00 g, 5.45 mmol) in 90% H₂SO₄ (15 mL)is warmed at 70° C. for 3.5 h, and then poured into ice-water. Themixture is extracted four times with EtOAc and the combined extractsworked up to give 6-chloro-3-nitropicolinamide (0.80 g, 73%). ¹H NMR(DMSO) δ 8.55 (1H, d, J=8.5 Hz), 8.31, 8.04 (1H, 1H, 2 brs), 7.93 (1H,d, J=8.5 Hz).

[0305] 6-Chloro-3H-pyrido[3,2-d)pyrimidin-4-one. A solution of6-chloro-3-nitropicolinamide (0.30 g, 1.49 mmol) in EtOAc (30 mL) ishydrogenated at 60 psi over 5% Pd-C (0.10 g) for 20 min. After removalof the catalyst by filtration the solution is concentrated to dryness togive 3-amino-6-chloropicolinamide as a yellow oil, which is useddirectly in the next step. It is dissolved in triethylorthoformate (30mL) and the mixture is heated under reflux for 18 h. Petroleum ether (30mL) is added to the cooled solution, and the resulting precipitate ofcrude 6-chloro-3H-pyrido[3,2-d)pyrimidin-4-one (0.27 g, 99%) is filteredoff and dried in a vacuum oven.

[0306] 4-(3-Bromoanilino)-6-chloropyrido[3,2-d]pyrimidine. A suspensionof the above quinazolone (0.20 g, 1.10 mmol) in POCl₃ (30 mL) is heatedunder reflux for 3 h, and then concentrated to dryness under reducedpressure. The residue is partitioned between CH₂CO₂ and saturated NaHCO₃solution, and the organic portion is worked up to give4,6-dichloropyrido[3,2-d]pyrimidine (0.16 g, 73%) as a tan solid, whichis used directly in the next step. A solution of the crudedichloropyridopyrimidine (0.16 g, 0.80 mmol) and 3-bromoaniline (0.17mL, 1.60 mmol) in propan-2-ol (25 mL) containing a trace of conc. HCl iswarmed at 50° C. for 30 min. The cooled mixture is poured into saturatedNaHCO₃ and extracted with EtOAc, and the extract is worked up andchromatographed on silica gel. Elution with EtOAc/petroleum ether (1:4)gives 3-bromoaniline, while EtOAc/petroleum ether (1:1) elutes4-(3-bromoanilino)-6-chloropyrido[3,2-d]pyrimidine (0.17 g, 63%). ¹H NMR(CDCl₃) δ 8.90 (1H, brs,) 8.84 (1H, s), 8.30 (1H, dd, J=2.1, 2.0 Hz)8.17 (1H, d, J=8.8 Hz), 7.82-7.78 (1H, m) 7.73 (1H, d, J=8.8 Hz),7.32-7.29, (2H, m).

EXAMPLE 6

[0307] 4-(3-Bromoanilino)-6-aminopyrido[3,2-d]pyrimidine

[0308] Reaction of 4-(3-bromoanilino)-6-fluoropyrido[3,2-d)pyrimidine(0.12 g, 0.38 mmol) (described in a previous experimental) with asaturated solution of ammonia in ethanol in a pressure vessel at 100° C.for 18 h gives 6-amino-4-(3-bromoanilino)pyrido[3,2-d]pyrimidine, (87mg, 72%) ¹H NMR (CDCl₃) δ 8.76 (1H, brs), 8.64 (1H, s), 8.23 (1H, brs),7.93 (1H, d, J=9.0 Hz), 7.81 (1H, dt, J_(d)=7.7 Hz, J_(t)=1.8 Hz),7.28-7.22 (2H, m), 7.00 (1H, d, J=9.0 Hz), 4.90 (2H, brs).

EXAMPLE 7

[0309] 4-(3-Bromoanilino)-6-methylaminopyrido[3,2-d]pyrimidine

[0310] Reaction of 4-(3-bromoanilino)-6-fluoropyrido[3,2-d)pyrimidine(50 mg, 0.16 mmol) (described in a previous experimental) withmethylamine hydrochloride (32 mg, 0.47 mmol) and triethylamine (70 μL,0.55 mmol) in ethanol (10 mL) in a pressure vessel at 100° C. for 18 hgives 6-methylamino-4-(3-bromoanilino)pyrido[3,2-d]pyrimidine (43 mg,81%). ¹H NMR (CDCl₃) δ 8.81 (1H, brs), 8.61 (1H, s), 8.19 (1H, t, J=1.8Hz), 7.86 (1H, d, J=9.1 Hz,), 7.83 (1H, dt, J_(d)=7.7 Hz, J_(t)=1.8 Hz),7.28-7.21 (2H, m), 6.92 (1H, d, J=9.1 Hz), 4.97 (1H, q, J=5.0 Hz), 3.13(3H, d, J=5.0 Hz).

EXAMPLE 8

[0311] 4-(3-Bromoanilino)-6-dimethylaminopyrido[3,2-d]pyrimidine.

[0312] A mixture of 4-(3-bromoanilino)-6-fluoropyrido[3,2-d)pyrimidine(0.15 g, 0.47 mmol) (described in a previous experimental),dimethylamine hydrochloride (0.11 g, 1.41 mmol) and triethylamine (0.23mL, 1.64 mmol) in EtOH (15 mL) is heated in a pressure vessel at 100° C.for 18 h. The solvent is removed under reduced pressure, and the residueis partitioned between EtOAc and water. The organic portion is workedup, and the residue chromatographed on silica gel. Elution withEtOAc/petroleum ether (1:1) gives foreruns, while EtOAc elutes off4-(3-bromoanilino)-6-dimethylaminopyrido[3,2-d]pyrimidine (0.14 g, 86%).¹H NMR (CDCl₃) δ 8.72 (1H, brs), 8.56 (1H, s), 8.17 (1H, t, J=1.9 Hz),7.85 (1H, d, J=9.3 Hz), 7.77 (1H, dt, J_(d)=7.5 Hz, J_(t)=1.9 Hz),7.27-7.18 (2H, m), 7.08 (1H., d, J=9.3 Hz), 3.21 (6H, s)

EXAMPLE 9

[0313] 4-(3-Bromoanilino)-6-methoxypyrido[3,2-d]pyrimidine

[0314] 4-(3-Bromoanilino)-6-fluoropyrido[3,2-d]pyrimidine (described ina previous experimental) (0.11 g, 0.34 mmol) is added to a solution ofNaOMe (prepared by the addition of Na metal (31 mg, 1.38 mmol) to dryMeOH (15 mL). After heating in a pressure vessel at 90° C. for 3 h, thesolution is concentrated to dryness and the residue is partitionedbetween EtOAc and water. Workup of the organic portion gives4-(3-bromophenyl)amino-6-methoxypyrido[3,2-d]pyrimidine (92 mg, 82%). ¹HNMR (CDCl₃) δ 8.73 (1H, s), 8.66 (1H, brs), 8.18 (1H, m), 8.05 (1H, d,J=8.9 Hz), 7.83-7.80 (1H, m), 7.30-7.24 (2H, m), 7.23 (1H, d, J=8.9 Hz),4.12 (3H, s)

EXAMPLE 10

[0315] 4-Anilinopyrido[4,3-d]pyrimidine

[0316] 4-(N-t-Butoxycarbonylamino)pyridine. To a mixture of4-aminopyridine (2-g, 21.24 mmol), potassium hydroxide (3.57 g, 63.72mmol), water (10 mL), and 2-methyl-2-propanol (4 mL) on ice is addeddi-t-butyl-dicarbonate (6.95 g, 31.87 mmol). The resulting biphasicsolution is stirred at 25° C. for 1 week, then water (20 mL) is added.The solution is extracted with 1X CH₂Cl₂ and 2X EtOAc. The organic layeris dried (MgSO₄) and concentrated under reduced pressure to give4-(N-t-butoxycarbonylamino)pyridine (4.08 g, 99%). ¹H NMR (DMSO) δ 9.84(1H, s), 8.35 (2H, d, J=6 Hz), 7.44 (2H, d, J=7 Hz), 1.49 (9H, s).

[0317] 4-(N-t-Butoxycarbonylamino)nicotinic acid. n-Butyl lithium (2.18M, 24 mL, 52.51 mmol) is added slowly to a solution of4-(N-t-butoxycarbonylamino)-pyridine (4.08 g, 21 mmol) in THF (50 mL,stirred under N₂ at −78° C. The solution is allowed to warm to 0° C.,stirred for 3 h, then cooled again to −78° C. and poured into ether (100mL) containing dry ice. The solution is warmed to room temperature withconstant stirring. Water is added and the mixture is neutralized withacetic acid. The resulting solid is collected by vacuum filtration anddried in a vacuum oven to give 4-(N-t-butoxycarbonylamino)nicotinic acid(2.72 g, 54%) as a brown solid. ¹H NMR (DMSO) δ 11.75 (1H, brs), 8.95(1H, s), 8.50 (1H, d, J=6.0 Hz), 8.20 (1H, d, J=6.0 Hz), 1.49 (9H, s)

[0318] 4-Amino nicotinic acid. A mixture of4-(N-t-butoxycarbonylamino)nicotinic acid (2.72 g, 11.4 mmol), TFA (10mL), and CH₂Cl₂ (20 mL) is stirred at room temperature for 12 h. Thevolatiles are removed under reduced pressure, and the resulting crude4-amino nicotinic acid is used directly in the next reaction.

[0319] 3H-Pyrido[4,3-d]pyrimidin-4-one. Crude 44-amino nicotinic acid(2.72 g, 11.4 mmol) in formamide (20 mL) is heated to 170° C. for 12 h.The volatiles are distilled out under reduced pressure (0.8 mmHg). Theresidual solid is then purified on a medium pressure silica gel column,eluting with 10% MeOH in CHCl₃ to give 3H-pyrido[4,3-d]pyrimidin-4-one(780 mg, 47%) as a whitish yellow solid. 1H NMR (DMSO) δ 12.64 (1H,brs), 9.28 (1H, s), 8.83 (1H, d, J=5.5 Hz), 8.30 (1H, s), 7.58 (1H, d,J=5.8 Hz).

[0320] 3H-Pyrido[4,3-d]pyrimidin-4-thione. Phosphorous pentasulfide(2.59 g, 5.83 mmol) is added to a solution of3H-pyrido[4,3-d]pyrimidin-4-one (780 mg, 5.3 mmol) in pyridine (5 mL).The mixture is refluxed for 5 h. On cooling a precipitate forms and thesupernatent is decanted off. The solid is suspended in water (20 mL) andthen filtered to yield 3H-pyrido[4,3-d]pyrimidin-4-thione (676 mg, 78%)as a black solid. ¹H NMR (DMSO) δ 14.53 (1H, brs), 9.65 (1H, s), 8.84(1H, d, J=7.0 Hz), 8.32 (1H, s), 7.64 (1H, d, J=8.0 Hz).

[0321] 4-Methylthiopyrido[4 3-d]pyrimidine. A mixture of3H-pyrido[4,3-d]pyrimidin-4-thione (676 mg, 4.14 mmol), triethylamine(1.4 mL, 10.31 mmol), DMSO (4 mL), and iodomethane (0.48 mL, 7.72 mmol)is stirred for 12 h under N₂ at 25° C. The mixture is poured onto waterand extracted with EtOAc. The organic extracts are dried (MgSO₄), andthe solvent is removed under reduced pressure to yield4-methylthiopyrido[4,3-d]pyrimidine (1.15 g, quant.) as a brown solid.¹H NMR (DMSO) δ 9.52 (1H, s), 9.16 (1H, s), 8.95 (1H, d, J=6 Hz), 7.86(1H, d, J=8 Hz), 2.75 (1H, s).

[0322] 4-Anilinopyrido[4,3-d]pyrimidine. A mixture of4-methylthiopyrido[4,3-d]pyrimidine (174 mg, 0.97 mmol), and aniline(186.2 mg, 1.99 mmol) in EtOH (2 mL) is refluxed under N₂ for 12 h.Cooling to 0° C. forms a solid which is filtered to yield4-anilinopyrido-[4,3-d]pyrimidine (34.5 mg, 16%). ¹H NMR (DMSO) δ 10.29(1H, brs), 9.86 (1H, s), 8.82 (1H, d, J=5.8 Hz), 8.72 (1H, s), 7.85 (2H,d, J=7.5 Hz), 7.66 (1H, d, J=5.5 Hz), 7.45 (2H, t, J=8.0 Hz), 7.23 (1H,t, J=7.3 Hz).

EXAMPLE 11

[0323] 4-(3-Bromoanilino)pyrido[4,3-d]pyrimidine

[0324] A mixture of 4-methylthiopyrido[4,3-d]pyrimidine (171 mg, 0.96mmol), (see previous experimental) and 3-bromoaniline (1 mL) is heatedto 100° C. for 2 h. A solid precipitates on cooling and is collected byvacuum filtration and then recrystallized from EtOH to yield4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (30 mg, 10%). ¹H NMR (DMSO) δ10.33 (1H, s), 9.86 (1H, s), 8.84 (1H, d, J=5.8 Hz), 8.79 (1H, s), 8.22(1H, s), 7.89 (1H, d, J=7.2 Hz), 7.69 (1H, d, J=5.8 Hz), 7.40 (2H, dt,J_(d)=8.0 Hz, J_(t)=1.5 Hz).

EXAMPLE 12

[0325] 4-(3-Bromoanilino)-7-fluoropyrido[4,3-d]pyrimidine

[0326] 3-Cyano-4,6-diaminopyridine. Crude2-bromo-3-cyano-4,6-diaminopyridine [W. J. Middleton, U.S. Pat. No.2,790,806 (Apr. 30, 1957), Du Pont; Chem. Abst. 51:P14829 (1957), seealso next experimental] (15.1 g, 0.071 mole) is hydrogenated in THF/MeOH(200 mL, 2:1) containing KOAc (7.0 g, 0.071 mole) and 5% Pd/C (4 g) at55 p.s.i. and 20° C. for 7 days. Filtration over celite, washing withTHF/MeOH and removal of the solvent gives a solid, which is dissolved indilute HCl and water. Adjustment of the solution pH to 10 (conc. NaOH)and cooling gives 3-cyano-4,6-diaminopyridine (6.58 g, 69%) as a yellowsolid, mp 197-198 IC (Metzger, R.; Oberdorfer, J.; Schwager, C.;Thielecke, W.; Boldt, P. Liebigs Ann. Chem. 1980, 946-953 record mp(benzene) 205° C. Extraction of the remaining liquor with EtOAc (4×200mL) gives further product (2.12 g, 22%). ¹H NMR (DMSO) δ 7.91 (1H, s),6.26, 6.24 (2H, 2H, brs), 5.63 (1H, s).

[0327] 4,6-Diamino-3-pyridylcarboxamide. 3-Cyano-4,6-diaminopyridine(4.30 g, 0.032 mole) is added to 90% H₂SO₄ (25 mL), then stirred at60-70° C. for 3 h. The resulting solution is added to cold conc. NaOH(40%) to give a mixture of 4,6-diamino-3-pyridylcarboxamide andinorganic salts. An analytically pure sample is obtained bychromatography on alumina (10-50% MeOH/CHCl₃) to give a pale yellowsolid. ¹H NMR (DMSO) δ 8.15 (1H, s), 6.91 (2H, brs), 7.7-6.3 (2H, brm),5.78 (2H, brs), 5.56 (1H, s).

[0328] 7-Amino-4-oxo-3H-pyrido-[4,3-d]pyrimidine. Crude4,6-diamino-3-pyridylcarboxamide (9.2 g) is heated in purified (EtO)₃CH(distilled from Na, 60 mL) at 170° C. for 1.5 d. After removing thesolvent, the residue is dissolved in hot 2 M NaOH, filtered, neutralized(conc. HCl) and cooled to give 7-amino-4-oxo-3H-pyrido[4,3-d]pyrimidine(3.57 g, 69% from the nitrile) as a light brown solid ¹H NMR (DMSO) δ11.79 (1H, brs), 8.74 (1H, s), 7.97 (1H, s), 6.76 (2H, brs), 6.38 (1H,s).

[0329] 7-Fluoro-4-oxo-3H-pyrido[4,3-d]-pyrimidine. A solution of7-amino-4-oxo-3H-pyrido[4,3-d]pyrimidine (1.00 g, 6.17 mmol) in 60% HBF₄(25 mL) at 0° C. is treated with solid NaNO₂ (0.85 g, 12.3 mmol, addedin portions over 2 h), and is then stirred at 0° C. for a further 1 hand at 20° C. for 30 min. The resulting mixture is ice-cooled,neutralized with saturated aqueous Na₂CO₃, and extracted with EtOAc(4×100 mL) The extract is washed with water, then filtered throughsilica gel (EtOAc) to give 7-fluoro-4-oxo-3H-pyrido[4,3-d]pyrimidine(0.48 g, 47%) as a cream solid. ¹H NMR (DMSO) δ 12.69 (1H, brs), 9.01(1H, s), 8.31 (1H, s), 7.34 (1H, s)

[0330] 4-(3-Bromoanilino)-7-fluoropyrido[4,3-d]pyrimidine. A suspensionof 7-fluoro-4-oxo-3H-pyrido[4,3-d]pyrimidine (0.23 g, 1.39 mmol) inPOCl₃ (10 mL) is stirred under reflux for 3.5 h, and is thenconcentrated under vacuum. The resulting oil is ice-cooled, diluted withCH₂Cl₂ (100 mL), saturated aqueous Na₂CO₃ (40 mL) and ice, and stirredat 20° C. for 2 h. The CH₂Cl₂ extract is separated and the aqueousportion further extracted with CH₂Cl₂ (2×100 mL), and then the combinedextracts are dried (Na₂SO₄) and filtered to give crude4-chloro-7-fluoropyrido[4,3-d]pyrimidine. 3-Bromoaniline (1.26 g, 7.35mmole), 3-bromoaniline hydrochloride (20 mg) and dry isopropanol (5 mL)are added, then the resulting solution is concentrated under vacuum toremove the CH₂Cl₂ and stirred at 20° C. for 1 h. Upon addition of diluteNaHCO₃ and water, the product crystallises. Filtration, washing withwater and CH₂Cl₂, gives pure4-(3-bromoanilino)-7-fluoropyrido[4,3-d]pyrimidine (297 mg, 67%) as acream solid. ¹H NMR (DMSO) δ 10.38 (1H, brs), 9.59 (1H, s), 8.72 (1H,s), 8.17 (1H, s), 7.85 (1H, m), 7.38 (3H, m)

EXAMPLE 13

[0331] 7-Amino-4-anilinopyrido[4,3-d]pyrimidine

[0332] 4,6-Diamino-2-bromo-3-cyanopyridine. HBr is bubbled for 2 h intoa mixture of malononitrile (16.3 g, 0.247 mol) and toluene (400 mL) at0° C. A light yellow precipitate forms. The reaction mixture is thenheated at 100° C. for 2 h, with much gas evolution. After cooling toroom temperature, the yellow solid is isolated via suction filtration,washed with toluene and air dried. The solid (25.96 g) is mixed withwater (500 mL), and the pH of the suspension is adjusted to 9-10 withNH₄OH (conc. ˜15 mL). After stirring at room temperature for 1 h, themixture is filtered. Recrystallization from EtOH affords a yellow solid.After-drying at 60° C. in a vacuum oven,4,6-diamino-2-bromo-3-cyanopyridine (12.95 g, 49%) is obtained. ¹H NMR(DMSO) δ 6.67 (2H,brs), 6.55 (2H, brs), 5.59 (1H, s)

[0333] 2,4-Diamino-5-cyanopyridinium acetate.4,6-Diamino-2-bromo-3-cyanopyridine (12.77 g, 60 mmol) is hydrogenatedin THF/MeOH (240 mL, 2:1) containing KOAc (5.9 g, 60 mmol) and 20% Pd/C(0.5 g) at 18 psi at 25° C. for 4 h. The mixture is celite filtered andthe solvent is stripped under reduced pressure to give a solid (11.15 g)which is stirred with THF (100 mL) at room temperature for 20 min. Themixture is refiltered and the filtrate is stripped to dryness to givethe desired product. After drying in a vacuum oven,2,4-diamino-5-cyanopyridinium acetate (10.65 g, 92%) is collected as ayellow solid. ¹H NMR (DMSO) δ 7.90 (1H, s), 6.26 (4H, brs), 5.62 (1H,s), 1.90 (3H, s).

[0334] 7-Amino-4-thiono-3H-pyrido[4,3-d]pyrimidine. A mixture of2,4-diamino-5-cyanopyridinium acetate (0.199 g, 1.0 mmol), triethylorthoformate (1.95 mL) and Ac₂O (1.95 mL) is refluxed under N₂ withstirring for 3 h. The solvent is then stripped and the residue isdissolved in MeOH (10 mL) containing NaOMe (0.81 g, 15 mmol). H₂S isbubbled through the mixture for ˜5 min, which is then refluxedovernight. After the solvent is stripped, the residue is dissolved inhot water and boiled with charcoal. After filtration, the filtrate isneutralized with acetic acid whilst hot to generate a yellow solid. Oncooling, the solid is collected by suction filtration, and is dried in avacuum oven overnight. 7-Amino-4-thiono-3H-pyrido[4,3-d]pyrimidine (84mg, 51%) is isolated as light yellow solid. ¹H NMR (DMSO) δ 9.82 (1H,s), 9.34 (1H, s), 8.37 (1H, s), 7.80 (2H, d, J=7.5 Hz), 7.38 (2H, t,J=7.5 Hz), 7.12 (1H, t, J=7.5 Hz), 6.61 (2H, brs) 6.43 (1H, s)

[0335] 7-Amino-4-methylthiopyrido[4,3-d]pyrimidine. NEt₃ (6 mL, 43 mmol)is added to a solution of 7-amino-4-thiono-3H-pyrido[4,3-d]pyrimidine(0.77 g, 4.3 mmol) in DMSO (7 mL) stirred under N₂ at 25° C. After thetwo phases have been stirred for 20 min, MeI (0.26 mL, 4.2 mmol) isadded. After 2 h, the reaction mixture is poured onto stirringice-water. Solid forms instantly. After further cooling at 0° C., thesolid is collected by suction filtration and dried in a vacuum oven togive 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (0.564 g, 68%). ¹H NMR(DMSO) δ 8.98 (1H, s), 8.71 (1H, s), 6.94 (2H, brs), 6.49 (1H, s) 2.63(3H, s).

[0336] 7-Amino-4-anilinopyrido[4,3-d]pyrimidine. A mixture of7-amino-4-methylthiopyrido[4,3-d]pyrimidine (0.136 g, 0.7 mmol) andaniline (0.5 mL, 5.5 mmol) is refluxed under N₂ at 180° C. for 2 h. Thereaction mixture is cooled to 25° C., when it precipitates. The solid iscollected by suction filtration and recrystallized from isopropanol, anddried in a vacuum oven overnight.7-Amino-4-anilinopyrido[4,3-d]pyrimidine (84 mg, 51%) is isolated as alight yellow solid. ¹H NMR (DMSO) δ 9.82 (1H, s), 9.34 (1H, s), 8.37(1H, s), 7.80 (2H, d, J=7.5 Hz), 7.38 (2H, t, J=7.5 Hz), 7.12 (1H, t,J=7.5 Hz), 6.61 (2H, brs) 6.43 (1H, s)

EXAMPLE 14

[0337] 7-Amino-4-(3-hydroxyanilino)pyrido[4,3-d]pyrimidine

[0338] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (299 mg,1.56 mmole) and 3-aminophenol (1.60 g, 14.7 mmole) is stirred at 160° C.for 15 min. The resulting product is chromatographed over silica gel (9%MeOH/CH₂Cl₂) to give 7-amino-4-(3-hydroxyanilino)pyrido[4,3-d]pyrimidine(108 mg, 18%) as a pale orange solid. ¹H NMR (DMSO) δ 9.69 (1H, brs),9.44 (1H, brs), 9.33 (1H, s), 8.38 (1H, s), 7.37 (1H, t, J=2.1 Hz), 7.21(1H, brd, J=8.4 Hz), 7.14 (1H, t, J=8.0 Hz), 6.59 (2H, brs), 6.53 (1H,ddd, J=7.9, 2.2, 0.8 Hz), 6.43 (1H, s)

EXAMPLE 15

[0339] 7-Amino-4-(3-methoxyanilino)pyrido[4,3-d]pyrimidine

[0340] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (226 mg,1.18 mmol) (described in the previous experimental) and m-anisidine(1.00 mL, 8.90 mmol) is stirred under N₂ at 190° C. for 1.5 h. Theresulting product is chromatographed over silica gel (5-7% EtOH/EtOAc)to give 7-amino-4-(3-methoxyanilino)pyrido[4,3-d]pyrimidine (136 mg,43%) as a light brown solid. ¹H NMR (DMSO) δ 9.78 (1H, brs), 9.34 (1H,s), 8.40 (1H, s), 7.50 (1H, brs), 7.44 (1H, d, J=8.0 Hz), 7.28 (1H, t,J=8.2 Hz), 6.71 (1H, dd, J=8.2, 2.3 Hz), 6.61 (2H, brs), 6.45 (1H, s),3.77 (3H, s).

EXAMPLE 16

[0341] 7-Amino-4-(2-methoxyanilino)pyrido[4,3-d]pyrimidine

[0342] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (227 mg,1.18 mmole) and o-anisidine (1.00 mL, 8.87 mmol) is stirred under N₂ at180° C. for 2.5 h. The resulting product is chromatographed over silicagel (5% EtOH/EtOAc) to give7-amino-4-(2-methoxyanilino)pyrido[4,3-d]pyrimidine (147 mg, 47%) as ayellow solid. ¹H NMR (DMSO) δ 9.44 (1H, brs), 9.25 (1H, s), 8.22 (1H,s), 7.54 (1H, dd, J=7.7, 1.4 Hz), 7.24 (1H, ddd, J=8.1, 7.4, 1.5 Hz),7.10 (1H, dd, J =8.2, 1.2 Hz), 6.98 (1H, dt, J_(d)=1.3 Hz, JC=7.5 Hz),6.52 (2H, brs), 6.41 (1H, s), 3.79 (3H, s)

EXAMPLE 17

[0343] 7-Amino-4-(3-aminoanilino)pyrido[4,3-d]pyrimidine

[0344] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (307 mg,1.60 mmol) (described in a previous experimental) and 3-nitroaniline(2.00 g, 14.5 mmol) is stirred at 200° C. for 1.5 h, and the crudeproduct is suspended in MeOH/THF (4:1, 250 mL) and hydrogenated over 5%Pd/C (2 g) at 60 psi and 20° C. for 24 h. The solution is filtered overcelite, washing thoroughly (hot MeOH), and is then absorbed onto aluminaand chromatographed on alumina (4-8% EtOH/CHCl₃) to give7-amino-4-(3-aminoanilino)pyrido[4,3-d]pyrimidine (66 mg, 16%) as agreen solid,. ¹H NMR (DMSO) δ 9.57 (1H, brs), 9.30 (1H, s), 8.33 (1H,s), 7.04 (1H, t, J=2.0 Hz), 6.99 (1H, t, J=8.0 Hz), 6.88 (1H, brd, J=8.0Hz), 6.55 (2H, brs), 6.40 (1H, s), 6.34 (1H, dd, J=7.9, 1.3 Hz), 5.10(2H, brs).

EXAMPLE 18

[0345] 7-Amino-4-(4-aminoanilino)pyrido[4,3-d]pyrimidine

[0346] 7-Amino-4-(4-acetamidoanilino)pyrido[4,3-d]pyrimidine. A mixtureof 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (138 mg, 0.72 mmole) and4-aminoacetanilide (1.50 g, 10.0 mmole) is stirred under N₂ at 200° C.for 1 h. The resulting product is chromatographed over alumina (8-10%MeOH/CH₂Cl₂) to give7-amino-4-(4-acetamidoanilino)pyrido[4,3-d]pyrimidine (110 mg, 52%) as apale yellow solid. ¹H NMR (DMSO) δ 9.94, 9.79 (1H, 1H, 2 brs), 9.31 (1H,s), 8.34 (1H, s), 7.69 (2H, d, J=8.9 Hz), 7.57 (2H, d, J=8.9 Hz), 6.57(2H, brs), 6.43 (1H, s), 2.05 (3H, s).

[0347] 7-Amino-4-(4-aminoanilino)pyrido[4,3-d]pyrimidine. A solution of7-amino-4-(4-acetamidoanilino)pyrido[4,3-d]pyrimidine (0.30 g, 1.02mmole) in aqueous NaOH (2 M, 10 mL) and MeOH (10 mL) is stirred at 100°C. for 7 h. The resulting product is chromatographed over alumina (3-4%EtOH/CHCl₃) to give 7-amino-4-(4-aminoanilino)pyrido[4,3-d]pyrimidine(86 mg, 33%) as an orange solid. ¹H NMR (DMSO) δ 9.58 (1H, brs), 9.24(1H, s), 8.25 (1H, s), 7.31 (2H d, J=8.6 Hz), 6.58 (2H, d, J=8.6 Hz),6.48 (2H, brs), 6.39 (1H, s), 5.00 (2H, brs).

EXAMPLE 19

[0348] 7-Amino-4-(3-dimethylaminoanilino)pyrido[4,3-d]pyrimidine

[0349] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (245 mg,1.28 mmol) (described in a previous experimental) andN,N-dimethyl-1,3-phenylenediamine (1.60 g, 11.8 mmol) is stirred underN₂ at 190° C. for 1 h, and the resulting product is chromatographed(twice) over alumina (3-EtOH/CHCl₃) to give7-amino-4-(3-dimethylaminoanilino)pyrido[4,3-d]pyrimidine (113 mg, 32%)as a pale yellow solid. ¹H NMR (DMSO) δ 9.66 (1H, brs), 9.33 (1H, s),8.36 (1H, s), 7.22 (1H, brd, J=7.8 Hz), 7.16 (2H, m), 6.57 (2H, brs),6.51 (1H, ddd, J=8.0, 2.3, 1.2 Hz), 6.42 (1H, s), 2:91 (6H, s).

EXAMPLE 20

[0350] 7-Amino-4-(4-dimethylaminoanilino)pyrido[4,3-d]pyrimidine

[0351] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (256 mg,1.33 mmole) and N,N-dimethyl-1,4-phenylenediamine (1.95 g, 14.4 mmole)is stirred under N₂ at 190° C. for 20 min. The resulting product ischromatographed over alumina (3-7% EtOH/CHCl₃) to give7-amino-4-(4-dimethylaminoanilino)pyrido[4,3-d]pyrimidine (198 mg, 53%)as an orange solid. ¹H NMR (DMSO) δ 9.67 (1H, brs), 9.27 (1H, s), 8.27(1H, s), 7.51 (2H, d, J=8.9 Hz), 6.75 (2H, d, J=8.9 Hz), 6.51 (2H, brs),6.39 (1H, s), 2.89 (6H, s).

EXAMPLE 21

[0352] 7-Amino-4-(2-nitroanilino)pyrido[4,3-d]pyrimidine

[0353] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (220 mg,1.15 mmole) and 2-nitroaniline (2.00 g, 14.5 mmole) is heated to 100°C., then excess dry HCl gas is added to the hot stirred solution, andthe mixture stirred at 160° C. for 20 min. The resulting product isneutralized with excess NaHCO₃, dissolved in MeOH/CHCl₃, dried ontosilica gel and chromatographed over silica gel (2-4% MeOH/CH₂Cl₂) togive 7-amino-4-(2-nitroanilino)pyrido[4,3-d]pyrimidine (108 mg, 33%) asa yellow brown solid. ¹H NMR (DMSO) δ 10.40 (1H, brs), 9.24 (1H, brs),8.20 (1H, brs), 8.12 (1H, brs), 8.01 (2H, brs), 7.75 (1H, brs), 6.70(2H, brs), 6.43 (1H, brs).

EXAMPLE 22

[0354] 7-Amino-4-(3-nitroanilino)pyrido[4,3-d]pyrimidine

[0355] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (127 mg,0.66 mmol) (described in a previous experimental) and 3-nitroaniline(1.70 g, 12.3 mmol) is stirred under N₂ at 200° C. for 1.5 h. Theresulting product is chromatographed over alumina (5-20% EtOH/CHCl₃) togive 7-amino-4-(3-nitroanilino)pyrido[4,3-d]pyrimidine (81 mg, 39%) as abrown solid. ¹H NMR (DMSO) δ 10.17 (1H, brs), 9.37 (1H, s), 8.87 (1H,brs), 8.48 (1H, s), 8.33 (1H, brd, J=7.5 Hz), 7.95 (1H, ddd, J=8.2, 2.1,1.0 Hz), 7.67 (1H, t, J=8.2 Hz), 6.70 (2H, brs), 6.47 (1H, s).

EXAMPLE 23

[0356] 7-Amino-4-(3-fluoroanilino)pyrido[4,3-d]pyrimidine

[0357] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (215 mg,1.12 mmol) and 3-fluoroaniline (1.16 g, 10.4 mmol) is stirred at 160° C.for 30 min. The resulting product is chromatographed over silica gel(6-7% MeOH/CH₂Cl₂) to give7-amino-4-(3-fluoroanilino)pyrido[4,3-d]pyrimidine (185 mg, 65%) as awhite solid. ¹H NMR (DMSO) δ 9.94 (1H, brs), 9.36 (1H, s), 8.46 (1H, s),7.91 (1H, brd, J=11.9 Hz), 7.63 (1H, brd, J=8.1 Hz), 7.41 (1H, dd,J=15.7, 7.7 Hz), 6.93 (1H, dt, J_(t)=8.5 Hz, J_(d)=2.4 Hz), 6.68 (2H,brs), 6.38 (1H, s)

EXAMPLE 24

[0358] 7-Amino-4-(3-chloroanilino)pyrido[4,3-d]pyrimidine

[0359] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (208 mg,1.08 mmol) and 3-chloroaniline (1.21 g, 9.48 mmol) is stirred at 150° C.for 20 min. The resulting product is chromatographed over alumina (5-10%MeOH/CH₂Cl₂) to give 7-amino-4-(3-chloroanilino)pyrido[4,3-d]pyrimidine(177 mg, 60%) as a white solid. ¹H NMR (DMSO) δ 9.92 (1H,brs), 9.35 (1H,s), 8.45 (1H, s), 8.08 (1H, brs), 7.79 (1H, brd, J=8.0 Hz), 7.40 (1H, t,J=8.1 Hz), 7.16 (1H, dd, J=7.9, 1.3 Hz), 6.68 (2H, brs), 6.46 (1H, s).

EXAMPLE 25

[0360] 7-Amino-4-(3,4-dichloroanilino)pyrido[4,3-d]pyrimidine

[0361] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (247 mg,1.29 mmol) and 3,4-dichloroaniline (1.50 g, 9.26 mmol) is stirred at165° C. for 30 min. The resulting product is chromatographed over silicagel (7-8% MeOH/CH₂Cl₂) to give7-amino-4-(3,4-dichloroanilino)pyrido[4,3-d]pyrimidine (252 mg, 64%) asa pale yellow solid. ¹H NMR (DMSO) δ 9.97 (1H, brs), 9.34 (1H, s), 8.47(1H, s), 8.29 (1H, brs), 7.86 (1H, brd, J=8.6 Hz), 7.62 (1H, d, J=8.8Hz), 6.70 (2H, brs), 6.46 (1H, s).

EXAMPLE 26

[0362] 7-Amino-4-(2-bromoanilino)pyrido[4,3-d]pyrimidine

[0363] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (198 mg,1.03 mmol) (described in a previous experimental) and 2-bromioaniline(1.00 mL, 9.18 mmol) is stirred under N₂ at 180° C. for 2.5 h, and theresulting product is chromatographed on alumina (1% EtOH/CHCl₃) to give7-amino-4-(2-bromoanilino)pyrido[4,3-d]pyrimidine (108 mg, 33%) as apale yellow solid, ¹H NMR (DMSO) δ 9.91 (1H, brs), 9.27 (1H, s), 8.20(1H, s), 7.73 (1H, d, J=7.9 Hz), 7.50 (1H, m), 7.44 (1H, t, J=6.9 Hz),7.25 (1H, m), 6.59 (2H, brs), 6.42 (1H, s).

EXAMPLE 27

[0364] 7-Amino-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine

[0365] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (167 mg,0.87 mmol) (described in a previous experimental) and 3-bromoaniline(0.75 mL, 7.8 mmol) is stirred under N₂ at 190° C. for 2.5 h, and theprecipitate which appears on cooling is recrystallized from Pr^(i)OH. ¹HNMR (DMSO) δ 9.91 (1H, brs), 9.34 (1H, s), 8.45 (1H, s), 8.19 (1H, s),7.84 (1H, d, J=8.0 Hz), 7.34 (1H, t, J=8.0 Hz), 7.29 (1H, d, J=8.2 Hz),6.68 (2H, brs), 6.45 (1H, s).

EXAMPLE 28

[0366] 7-Amino-4-(4-bromoanilino)pyrido[4,3-d]pyrimidine

[0367] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (261 mg,1.36 mmole) and 4-bromoaniline (1.00 g, 5.81 mmole) is stirred under N₂at 200° C. for 15 min. The resulting product is chromatographed onsilica gel (10-15% EtOH/EtOAc) to give7-amino-4-(4-bromoanilino)pyrido[4,3-d]pyrimidine (200 mg, 46%) as apale yellow solid. ¹H NMR (DMSO) δ 9.88 (1H, brs) 9.34 (1H, s), 8.40(1H, s), 7.83 (2H, d, J=8.8 Hz), 7.55 (2H, d, J=8.8 Hz), 6.64 (2H, brs),6.44 (1H, s).

EXAMPLE 29

[0368] 7-Amino-4-(3-iodoanilino)pyrido[4,3-d]pyrimidine

[0369] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (72 mg,0.37 mmol) and 3-iodoaniline (1.25 g, 5.71 mmol) is stirred at 160° C.for 30 min. The resulting product is chromatographed over silica gel(5-7% MeOH/CH₂Cl₂) to give7-amino-4-(3-iodoanilino)pyrido[4,3-d]pyrimidine (83 mg, 61) as a lightbrown rosettes. ¹H NMR (DMSO) δ 9.84 (1H, brs), 9.34 (1H, s), 8.44 (1H,s), 8.30 (1H, brs), 7.90 (1H, dd, J=7.9, 0.8 Hz), 7.47 (1H, d, J=7.7Hz), 7.18 (1H, t, J=8.0 Hz), 6.66 (2H, brs), 6.46 (1H, s).

EXAMPLE 30

[0370] 7-Amino-4-(2-trifluoromethylanilino)pyrido[4,3-d]pyrimidine

[0371] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (300 mg,1.56 mmol), 2-aminobenzotrifluoride hydrochloride (1.00 g, 5.06 mmol)and 2-aminobenzotrifluoride (2.00 g, 12.4 mmol) is stirred at 160° C.for 10 min. The resulting product is neutralized with excess NaHCO₃,dissolved in MeOH/CHCl₃, dried onto silica gel and chromatographed oversilica gel (6-7% MeOH/CH₂Cl₂) to give7-amino-4-(2-trifluoro-methylanilino)pyrido[4,3-d]pyrimidine (194 mg,41%) as a cream solid, mp (MeOH/CHCl₃/light petroleum) 126-130° C.(dec.). ¹H NMR (DMSO) δ 10.60 (1H, brs), 9.17 (1H, brs), 8.13 (1H, brs),7.76, 7.69 (1H, 1H, m, m), 7.45 (2H, m), 6.66 (2H, brs), 6.36 (1H, s).

EXAMPLE 31

[0372] 7-Amino-4-(3-trifluoromethylanilino)pyrido[4,3-d]pyrimidine

[0373] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (234 mg,1.22 mmol) (described in a previous experimental) and3-aminobenzotrifluoride (2.00 mL, 16.0 mmol) is stirred under N₂ at190-200° C. for 2 h, and the resulting product is then chromatographedover silica gel (5-10% EtOH/EtOAc), and then over alumina (5-7%EtOH/CHCl₃) to give7-amino-4-(3-trifluoromethylanilino)pyrido[4,3-d]pyrimidine (157 mg,42%) as a cream solid. ¹H NMR (DMSO) δ 10.04 (1H, s), 9.37 (1H, s), 8.46(1H, s), 8.31 (1H, s), 8.19 (1H, d, J=8.2 Hz), 7.62 (1H, t, J=8.0 Hz),7.45 (1H, d, J=7.7 Hz), 6.69 (2H, brs), 6.47 (1H, s).

EXAMPLE 32

[0374] 7-Amino-4-(4-trifluoromethylanilino)pyrido[4,3-d]pyrimidine

[0375] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (390 mg,2.03 mmol), 4-aminobenzotrifluoride hydrochloride (0.40 g, 2.02 mmol)and 4-aminobenzotrifluoride (1.61 g, 10.0 mmol) is stirred at 180° C.for 2 min. The resulting product is neutralized with excess NaHCO₃,dissolved in MeOH/CHCl₃, dried onto alumina and chromatographed overalumina (4-7% MeOH/CH₂Cl₂) to give7-amino-4-(4-trifluoromethylanilino)pyrido[4,3-d]pyrimidine (390 mg,63%) as a cream solid. Analytically pure material was obtained byfurther chromatography over silica gel (5% MeOH/CH₂Cl₂) to give paleyellow needles. ¹H NMR (DMSO) δ 10.09 (1H, brs), 9.40 (1H, s), 8.48 (1H,s), 8.13 (2H, d, J=8.2 Hz), 7.74 (2H, d, J=8.7 Hz), 6.72 (2H, brs), 6.40(1H, s).

EXAMPLE 33

[0376] 4-(3-Bromoanilino)-7-methylaminopyrido[4,3-d]pyrimidine

[0377] A mixture of 7-fluoro-4-(3-bromoanilino)-pyrido[4,3-d]pyrimidine(74 mg, 0.23 mmol), triethylamine (7 mL, 50 mmol) and methylaminehydrochloride (3.0 g, 44 mmol) in isopropanol (30 mL) contained in asteel bomb is stirred at 95° C. (oil bath) for 5 h. The resultingmixture is concentrated under vacuum, basified with aqueous Na₂CO₃,diluted with water and extracted with EtOAc (3×100 mL). Chromatographyof this extract on silica gel (3% MeOH/CH₂Cl₂) gives4-(3-bromoanilino)-7-methylaminopyrido[4,3-d]pyrimidine (50 mg, 65%) asa pale yellow solid. ¹H NMR (DMSO) δ 9.93 (1H, brs), 9.37 (1H, s), 8.47(1H, s), 8.18 (1H, s), 7.84 (1H, d, J=7.8 Hz), 7.34 (1H, t, J=7.9 Hz),7.30 (1H, brd, J=8.1 Hz), 7.19 (1H, q, J=4.7 Hz), 6.35 (1H, s), 2.85(3H, d, J=4.8 Hz)

EXAMPLE 34

[0378] 4-(3-Bromoanilino)-7-dimethylaminopyrido[4, 3-d]pyrimidine

[0379] A mixture of 7-fluoro-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine(101 mg, 0.32 mmol), triethylamine (4.4 mL, 32 mmole) and dimethylaminehydrochloride (2.58 g, 32 mmol) in isopropanol (30 mL) contained in asteel bomb is stirred at 100° C. (oil bath) for 4 h. The resultingsolution is concentrated under vacuum, basified with aqueous Na₂CO₃ anddiluted with water to give a solid. Filtration and recrystallisationfrom MeOH/CHCl₃ gives7-dimethylamino-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine (102 mg, 94%)as a pale yellow solid. ¹H NMR (DMSO) δ 9.93 (1H, brs), 9.42 (1H, s),8.48 (1H, s), 8.19 (1H, s), 7.85 (1H, d, J=7.7 Hz), 7:35 (1H, t, J=7.9Hz), 7.30 (1H, brd, J=7.8 Hz), 6.53 (1H, s), 3.16 (6H, s).

EXAMPLE 35

[0380]4-[N-(3-Bromophenyl)-N-methylamino]-7-methylamino-pyrido[4,3-d]pyrimidine

[0381] A mixture of 7-fluoro-4-(3-bromoanilino)-pyrido[4,3-d]pyrimidine(100 mg, 0.31 mmole), triethylamine (4.4 mL, 32 mmole) and methylaminehydrochloride (2.12 g, 32 mmole) in isopropanol (30 mL) contained in asteel bomb is stirred at 100° C. (oil bath) for 5 h. The resultingmixture is concentrated under vacuum, basified with aqueous Na₂CO₃,diluted with water and extracted with EtOAc (3×100 mL). Chromatographyof this extract on silica gel (1-2% MeOH/CH₂Cl₂) gives4-[N-(3-bromophenyl)-N-methylamino]-7-methylaminopyrido[4,3-d]pyrimidine(23 mg, 21%) as a pale yellow solid. ¹H NMR (DMSO) δ 8.14 (1H, s), 7.79(1H, s), 7.30 (1H, t, J=8.0 Hz), 7.20 (1H, ddd, J=7.9, 1.8, 0.8 Hz),7.03 (1H, brq, J=4.9 Hz), 7.01 (1H, t, J=1.9 Hz), 6.82 (1H, ddd, J=7.8,1.8, 0.9 Hz), 6.25 (1H, s), 3.40 (3H, s), 2.73 (3H, d, J=4.9 Hz).

EXAMPLE 36

[0382] 7-Acetylamino-4-(3-bromoanilino)pyrido[4,3-d]pyrimidine

[0383] A mixture of 7-amino-4-(3-bromoanilino)-pyrido[4,3-d]pyrimidine(0.154 g, 0.49 mmol), acetic anhydride (0.14 mL, 1.5 mmol),triethylamine (0.14 mL, 1.0 mmol) and a catalytic amount of4-(N,N-dimethylamino)pyridine are stirred under N₂ at room temperaturefor 18 h. The reaction is then quenched by addition of ice water. Thedark precipitate is collected by Buchner filtration and is purified bypreparative tlc (Rf=0.25, 7% MeOH/CHCl₃). Recrystallization from EtOHgives 7-acetylamino-4-(3-bromoanilino)pyrido[4,3-d]-pyrimidine (13.5 mg,7.7%). ¹H NMR (DMSO) δ 10.92 (1H, s), 10.22 (1H, s), 9.64. (1H, s), 8.70(1H, s), 8.28 (1H, s), 8.21 (1H,s), 7.88 (1H, d, J=7.7Hz) 7.41-7.34 (3H,m), 2.16 (3H, s).

EXAMPLE 37

[0384] 4-(3-Bromoanilino)-7-methoxypyrido[4,3-d]pyrimidine

[0385] A solution of 7-fluoro-4-(3-bromoanilino)-pyrido[4,3-d]pyrimidine(100 mg, 0.31 mmol) in 1 M sodium methoxide-methanol (30 mL) is stirredunder reflux for 42 h. The resulting mixture is concentrated underreduced pressure, diluted with water and neutralized with dilute HCl togive 7-methoxy-4-(3-bromo-anilino)pyrido[4,3-d]pyrimidine (92 mg, 89%)as a white solid. ¹H NMR (DMSO) δ 10.22 (1H, brs), 9.57 (1H, s), 8.63(1H, s), 8.19 (1H, s), 7.86 (1H, brd, J=7.9 Hz), 7.39 (1H, t, J=7.9 Hz),7.35 (1H, dd, J=7.9, 1.5 Hz), 6.96 (1H, s), 4.00 (3H, s).

EXAMPLE 38

[0386] 4-Benzylaminopyrido[4,3-d]pyrimidine

[0387] 4-Methylthiopyrido[4,3-d]pyrimidine (160.4 mg, 0.902 mmol), andbenzylamine (106.3 mg, 0.992 mmol) in EtOH (2 mL) are heated at 80° C.for 12 h, and then the solvent is removed under reduced pressure. Theresulting solid is suspended in CH₂Cl₂, filtered, and the resultingsolid is purified by preparative tlc on silica, eluting with 5% MeOH inCHCl₃. Removal of the solvent under reduced pressure yields4-benzylaminopyrido[4,3-d]pyrimidine (36 mg, 17%). ¹H NMR (DMSO) δ 9.60(1H, s), 9.37 (1H, t, J=5.8 Hz), 8.72 (1H, d, J=5.8 Hz), 8.57 (1H, s),7.54 (1H, d, J=5.8 Hz), 7.37 (2H, d, J=7.0 Hz), 7.33 (2H, t, J=7.3 Hz),7.25 (1H, t, J=7.2 Hz), 4.81 (2H, d, J=5.8 Hz).

EXAMPLE 39

[0388] 4-([R]-1-Phenylethylamino)-pyrido[4,3-d]pyrimidine

[0389] To a mixture of 4-methylthiopyrido[4,3-d] pyrimidine (85 mg, 0.48mmol) and EtOH (2.5 mL) is added R-methylbenzylamine (0.13 mL, 1.0 mmol)dropwise. The resulting mixture is refluxed at 80° C. for 20 h. Thesolvent is removed under reduced pressure to give an oil which iscrystallized from MeOH to give4-([R]-1-phenylethylamino)pyrido[4,3-d]pyrimidine (41.6 mg, 35%), mp138-138.5° C. ¹H NMR (DMSO) δ 9.77 (1H, d, J=0.7 Hz), 9.00 (1H, d, J=7.7Hz), 8.73 (1H, d, J=5.8 Hz), 8.54 (1H, s), 7.53 (1H, dd, J=5.8, 0.5 Hz),7.45 (2H, .d, J=7.2 Hz), 7.33 (2H, t, J=7.6 Hz), 7.23 (1H, tt, J=7.5,1.2 Hz), 5.63 (1H, p, J=7.2 Hz), 1.61 (3H, t, J=7.0 Hz).

EXAMPLE 40

[0390] 7-Amino-4-benzylaminopyrido[4,3-d]pyrimidine

[0391] A mixture of 2,4-diamino,5-cyanopyridinium acetate (8.78 g, 45mmol), formic acid (10.66 g, 0.204 mol) and benzylamine (45 mL, 0.41mol) is heated at 200° C. under N₂ for 2 h. Upon cooling, it solidifies.Water (500 mL) is added and the gummy solid/water mixture is stirred for−20 min. at 0° C. The liquid is decanted. The solid is washed with waterand then recrystallized from isopropanol (25 mL). After drying in avacuum oven overnight, 7-amino-2-benzylaminopyrido[4,3-d]pyrimidine(8.29 g, 73%) is obtained as a light yellow solid. ¹H NMR (DMSO) δ 9.10(1H, s), 8.85 (1H, t, J=5.8 Hz), 8.25 (1H, s), 7.21-7.36 (5H, m), 6.46(2H, brs), 6.35 (1H, s), 4.74 (2H, d, J=6.0 Hz).

EXAMPLE 41

[0392] 7-Amino-4-([R]-1-thenylethylamino)pyrido[4,3-d]pyrimidine

[0393] A mixture of [R]-1-phenylethylamine (0.072 mL, 0.55 mmol) and7-amino-4-methylthiopyrido[4,3-d]pyrimidine (97 mg, 0.5 mmol) (describedin a previous experimental) is heated at 180° C. under N₂ for 1.5 hr.The reaction is then cooled to room temperature producing a precipitate.The mixture is added to water and CHCl₃, sonicated and filtered. Thephases are separated and the aqueous phase is extracted with CHCl₃. Thecombined extracts are washed with water, saturated brine and dried(MgSO₄). The solvent is removed under reduced pressure and the residuepurified by using preparative tic (5% MeOH/CHCl₃) and recrystallizationfrom CHCl₃ to give7-amino-4-([R]-1-phenylethylamino)pyrido[4,3-d]pyrimidine (14.5 mg,11%), mp 231.8-232.1° C. ¹H NMR (DMSO) δ 9.23 (1H, s), 8.50 (1H, d,J=8.0 Hz), 8.19 (1H, s), 7.41 (2H, d, J=7.0 Hz), 7.31 (2H, t, J=8.0 Hz,7.21 (1H, tt, J=7.4, 1.2 Hz), 6.45 (2H, s) 6.33 (1H, s), 5.56 (1H, p,J=7.2 Hz), 1.55 (3H, d, J=7.0 Hz).

EXAMPLE 42

[0394] 7-Amino-4-(2-aminobenzylamino)pyrido[4,3-d]pyrimidine

[0395] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (136 mg,0.71 mmol) (described in a previous experimental) and 2-aminobenzylamine(1.70 g, 13.8 mmol) in isopropanol (5 mL) is stirred at reflux for 1 h,and the resulting product is chromatographed on silica gel (7-20%EtOH/EtOAc) and alumina (6-100 EtOH/CHCl₃) to give7-amino-4-(2-aminobenzylamino)pyrido[4,3-d]pyrimidine (89 mg, 47%) as awhite solid. ¹H NMR (DMSO) δ 9.08 (1H, s), 8.68 (1H, t, J=5.8 Hz), 8.26(1H, s), 7.05 (1H, d, J=7.4 Hz), 6.96 (1H, t, J=7.6 Hz), 6.63 (1H, d,J=7.9 Hz), 6.51 (1H, t, J=7.4 Hz), 6.46 (2H, brs), 6.35 (1H, s), 5.20 (2H, brs), 4.56 (2H, d, J=5.8 Hz).

EXAMPLE 43

[0396] 7-Amino-4-(3-dimethylaminobenzylamino)pyrido[4,3-d]pyrimidine

[0397] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (236 mg,1.23 mmol) (described in a previous experimental) and3-dimethylamino-benzylamine (1.36 g, 9.07 mmol) in isopropanol (5 mL) isstirred under N₂ at reflux for 1 h, and the resulting product ischromatographed on silica gel (10-15% EtOH/EtOAc), then on alumina(1-EtOH/CHCl₃) to give7-amino-4-(3-dimethylaminobenzylamino)pyrido[4,3-d]pyrimidine (145 mg,40%) as a white solid. ¹H NMR (DMSO) δ 9.11 (1H, s), 8.79 (1H, t, J=5.9Hz), 8.26 (1H, s), 7.11 (1H, dd, J=8.0, 7.7 Hz), 6.73 (1H, brs), 6.63(1H, d, J=7.6 Hz), 6.60 (1H, dd, J=8.1, 2.2 Hz), 6.44 (2H, brs), 6.35(1H, s), 4.67 (2H, d, J=5.8 Hz), 2.86 (6H, s).

EXAMPLE 44

[0398] 7-Amino-4-(3-nitrobenzylamino)-pyrido[4,3-d]pyrimidine

[0399] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (228 mg,1.19 mmol) (described in a previous experimental) and 3-nitrobenzylamine(0.81 g, 5.33 mmol) is stirred under N₂ at 150-160° C. for 1.5 h, andthe resulting product chromatographed on silica gel (5-10% EtOH/EtOAc)to give 7-amino-4-(3-nitrobenzylmino)pyrido[4,3-d]pyrimidine (151 mg,43%) as a yellow solid. ¹H NMR (DMSO) δ 9.11 (1H, s), 8.98 (1H, t, J=5.5Hz), 8.26 (1H, s), 8.22 (1H, brs), 8.12 (1H, dd, J=8.0, 1.8 Hz), 7.83(1H, d, J=7.7 Hz), 7.63 (1H, t, J=7.9 Hz), 6.50 (2H, brs), 6.38 (1H, s),4.85 (2H, d, J=5.8 Hz).

EXAMPLE 45

[0400] 7-Amino-4-(3-methoxybenzylamino)pyrido[4,3-d]pyrimidine

[0401] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (136 mg,0.71 mmol) (described in a previous experimental) and3-methoxybenzylamine (1.37 g, 10.0 mmol) in isopropanol (3 mL) isstirred under N₂ at reflux for 3 h. Evaporation of the solvent andchromatography on silica gel (5-10% EtOH/EtOAc) gives7-amino-4-(3-methoxybenzylamino)pyrido[4,3-d]pyrimidine (153 mg, 77%) asa white solid. ¹H NMR (DMSO) δ 9.11 (1H, s), 8.83 (1H, t, J=5.7 Hz),8.26 (1H, s), 7.24 (1H, dt, J_(d)=0.8 Hz, J_(t)=8.1 Hz), 6.92 (2H, m),6:81 (1H, dt, J_(d)=8.2 Hz, J_(t)=1.2 Hz), 6.46 (2H, brs), 6.37 (1H, s),4.71 (2H, d, J=5.8 Hz), 3.73 (3H, s).

EXAMPLE 46

[0402] 7-Amino-4-(4-chlorobenzylamino)-pyrido[4,3-d]pyrimidine mesylate

[0403] The free base (56 mg, 0.20 mmol)(prepared from2,4-diamino,5-cyanopyridinium acetate, formic acid and4-chlorobenzylamine at 200° C. as described in a previous example isprecipitated from acetone solution with methanesulfonic acid (105 μL,0.23 mmol) to give a polymesylate salt. ¹H NMR (DMSO) δ 10.59 (1H, t,J=5.6 Hz), 9.24 (1H, s), 8.69 (1H, s), 7.42 (4H, s), 6.42 (1H, s), 5.8(˜6H, vbrs), 4.89 (2H, d, J=5.8 Hz), 2.41 (˜7.5H, s)

EXAMPLE 47

[0404] 7-Amino-4-(2-bromobenzylamino)pyrido[4,3-d]pyrimidine

[0405] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (225 mg,1.17 mmol) (described in a previous experimental) and 2-bromobenzylamine(0.84 g, 4.52 mmol) is stirred under N₂ at 140° C. for 1 h, and theresulting product chromatographed on silica gel (1-5% EtOH/EtOAc) togive 7-amino-4-(2-bromobenzylamino)pyrido[4,3-d]pyrimidine (175 mg, 45%)as a light brown solid. ¹H NMR (DMSO) δ 9.16 (1H, s), 8.85 (1H, t, J=5.7Hz), 8.24 (1H, s), 7.64 (1H, d, J=7.8 Hz), 7.34 (1H, dd, J=7.7, 7.1 Hz),7.31 (1H, dd, J=7.7, 2.4 Hz), 7.21 (1H, ddd, J=7.8, 6.9, 2.4 Hz), 6.50(2H, brs), 6.39 (1H, s), 4.74 (2H, d, J=5.7 Hz).

EXAMPLE 48

[0406] 7-Amino-4-(3-bromobenzylamino)pyrido[4,3-d]pyrimidine

[0407] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (228 mg,1.19 mmol) (described in a previous experimental) and 3-bromobenzylamine(0.84 g, 4.52 mmol) is stirred under N₂ at 140° C. for 1 h. Theresulting product is chromatographed on silica gel (2-10% EtOH/EtOAc) togive 7-amino-4-[(3-bromophenyl)methylamino]pyrido[4,3-d]pyrimidine (203mg, 52%) as a light brown solid. ¹H NMR (DMSO) δ 9.09 (1H, s), 8.86 (1H,t, J=5.8 Hz), 8.26 (1H, s), 7.54 (1H, s), 7.44 (1H, d, J=7.8 Hz,), 7.36(1H, d, J=7.6 Hz), 7.29 (1H, t, J=7.7 Hz), 6.48 (2H, s), 6.37 (1H, s),4.73 (2H, d, J=5.8 Hz).

EXAMPLE 49

[0408] 7-Amino-4-(4-bromobenzylamino)pyrido[4,3-d]pyrimidine

[0409] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (234 mg,1.22 mmol) (described in a previous experimental) and 4-bromobenzylamine(0.84 g, 4.52 mmol) is stirred under N₂ at 140° C. for 1 h, and theresulting product chromatographed on silica gel (10% EtOH/EtOAc) to give7-amino-4-(4-bromobenzylamino)pyrido[4,3-d]pyrimidine (192 mg, 48%) as acream solid. ¹H NMR (DMSO) δ 9.09 (1H, s), 8.87 (1H, t, J=5.7 Hz), 8.25(1H, s), 7.51 (2H, d, J=8.3 Hz), 7.31 (2H, d, J=8.3 Hz), 6.46 (2H, brs),6.37 (1H, s), 4.70 (2H, d, J=5.8 Hz).

EXAMPLE 50

[0410] 7-Amino-4-(2-trifluoromethylbenzylamino)pyrido[4,3-d]pyrimidine

[0411] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (225 mg,1.17 mmol) and 2-(trifluoromethyl)benzylamine (0.90 mL, 6.42 mmol) isstirred under N₂ at 150° C. for 1 h. The resulting product ischromatographed on silica gel (5% EtOH/EtOAc) to give7-amino-4-(2-trifluoromethylbenzyl) aminopyrido[4,3-d]pyrimidine (0.22g, 59%) as a white solid. ¹H NMR (DMSO) δ 9.16 (1H, s), 8.88 (1H, t,J=5.7 Hz), 8.23 (1H, s), 7.75 (1H, d, J=7.7 Hz), 7.62 (1H, t, J=7.5 Hz),7.50 (1H, d, J=7.4 Hz), 7.47 (1H, t, J=7.6 Hz), 6.51 (2H, brs), 6.39(1H, s), 4.92 (2H, d, J=5.5 Hz).

EXAMPLE 51

[0412] 7-Amino-4-(3-trifluoromethylbenzylamino)pyrido[4,3-d]pyrimidine

[0413] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (225 mg,1.17 mmole) and 3-(trifluoromethyl)benzylamine (0.63 mL, 4.40 mmole) isstirred under N₂ at 140° C. for 1 h. The resulting product ischromatographed on silica gel (3-5% EtOH/EtOAc) to give7-amino-4-[(3-trifluoromethylphenyl)methylamino]pyrido[4,3-d]pyrimidine(0.24 g, 63%) as a light brown solid. ¹H NMR (DMSO) δ 9.10 (1H, s), 8.92(1H, t, J=5.7 Hz), 8.26 (1H, s), 7.71 (1H, s), 7.66 (1H, d, J=7.4 Hz),7.62 (1H, d, J=7.8 Hz), 7.57 (1H, t, J=7.6 Hz), 6.49 (2H, brs), 6.38(1H, s), 4.82 (2H, d, J=5.8 Hz).

EXAMPLE 52

[0414] 7-Amino-4-(4-trifluoromethylbenzylamino)pyrido[4,3-d]pyrimidine

[0415] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (225 mg,1.17 mmol) and 4-(trifluoromethyl)benzylamine (0.63 mL, 4.42 mmol) isstirred under N₂ at 140° C. for 1 h. The resulting product ischromatographed on alumina (5-10% EtOH/CHCl₃) then silica gel (2-10%EtOH/EtOAc) to give7-amino-4-[(4-trifluoromethylphenyl)methylamino]pyrido[4,3-d]pyrimidine(0.21 g, 56%) as a light brown solid. ¹H NMR (DMSO) δ 9.12 (1H, s), 8.94(1H, t, J=5.8 Hz), 8.24 (1H, s), 7.69 (2H, d, J=8.1 Hz), 7.56 (2H, d,J=8.1 Hz), 6.48 (2H, brs), 6.38 (1H, s), 4.82 (2H, d, J=5.8 Hz).

EXAMPLE 53

[0416] 7-Amino-4-(thien-2-ylmethylamino)[4,3-d]pyrimidine dimesylate

[0417] The compound is obtained from 2,4-diamino,5-cyanopyridiniumacetate (190 mg, 0.98 mmol), formic acid (0.23 g, 4.4 mmol) andthienylmethylamine (1.07 ml, 10 mmol) as described in a previousexperimental. The crude product is converted into a dimesylate salt asdescribed previously and recrystallized from Pr^(i)OH to give7-amino-4-(thien-2-ylmethylamino)pyrido[4,3-d]pyrimidine dimesylate in19% yield. 1H NMR (DMSO δ 10.67 (1H, t, J=5.8 Hz), 9.21 (1H, s), 8.77(1H, s) 7-48 (1H, dd, J=5.1, 1.2 Hz) 7.16 (1H, dd, J=3.4, 0.7 Hz), 7:02(1H, dd, J=4.8, 3.4 Hz), 6.42 (1H, s) 5.06 (2H, d, J=5.7 Hz), 2.41 (6H,s).

EXAMPLE 54

[0418] 7-Acetylamino-4-benzylaminopyrido[4,3-d]pyrimidine

[0419] 7-Acetylamino-4-methylthiopyrido[4,3-d]pyrimidine. Acetylchloride (0.70 mL, 9.84 mmol) is added to a solution of7-amino-4-methylthiopyrido[4,3-d]pyrimidine (0.20 g, 1.04 mmol)(described in a previous experimental) and Et₃N (1.51 mL, 10.8 mmol) inTHF at 0° C., and then the mixture is stirred at 20° C. for 4 h. Water(50 mL) was added, then the solution was extracted with EtOAc (3×50 mL).Evaporation and chromatography on alumina (1% EtOH/CHCl₃) yields7-acetylamino-4-methylthiopyrido[4,3-d]pyrimidine (0.12 g, 49%) as ayellow solid,. ¹H NMR (DMSO) δ 11.05 (1H, s), 9.30 (1H, s), 9.02 (1H,s), 8.38 (1H, s), 2.71 (3H, s), 2.18 (3H, s).

[0420] 7-Acetylamino-4-benzylaminopyrido[4,3-d]pyrimidine. A mixture of7-acetylamino-4-methylthiopyrido[4,3-d]pyrimidine (0.40 g, 1.71 mmol)and benzylamine (1.0 mL, 9.15 mmol) is stirred under N₂ at 140° C. for 1h, and the resulting product is chromatographed on silica gel (EtOAc) togive 7-acetylamino-4-benzylaminopyrido[4,3-d]pyrimidine (0.31 g, 62%) asa white solid. ¹H NMR (DMSO) δ 10.79 (1H, s), 9.42 (1H, s), 9.23 (1H, t,J=5.8 Hz), 8.49 (1H, s), 8.18 (1H, s), 7.39 (1H, dt, J_(d)=6.9 Hz,J_(t)=1.7 Hz), 7.34 (1H, tt, J=7.3, 1.7 Hz), 7.25 (1H, tt, J=7.1, 1.7Hz), 4.80 (2H, d, J=5.8 Hz), 2.15 (3H, s)

EXAMPLE 55

[0421] 4-Anilinopyrido[3,4-d]pyrimidine

[0422] 4-Carboxamidonicotinic acid. 3,4-Pyridine dicarboxylic anhydride(8.3 g, 55.6 mmol) is added to conc NH₄OH (12 mL) in H₂O (60 mL) stirredat 0° C. over 5 min. Upon addition a paste forms which is stirred for 1h at room temperature. The white paste is sparged with N₂ for 30 min anddiluted with H₂O (10 mL) to form a clear solution. Then SO₂ is bubbledthrough the solution for 15 min reducing its pH to 2. Upon cooling theresulting solid is filtered, rinsed with H₂O, and oven dried to yield4-carboxamidonicotinic acid (7 g, 76%) as a white solid. ¹H NMR (DMSO) δ8.93 (1H, s), 8.76 (1H, d, J=5.0 Hz), 8.08 (1H, s), 7.62 (1H, s), 7.45(1H, d, J=5.0 Hz).

[0423] Isoquinolinic imide. 4-Carboxamidonicotinic acid (280 mg, 1.68mmol) is heated neat at 200° C. for 5 h to yield isoquinolinic imide(177.2 mg, 71%) as a white solid. ¹H NMR (DMSO) δ 11.68 (1H, s),9.12-9.03 (2H, m), 7.80 (1H, d, J=5.1 Hz).

[0424] 3-Amino isonicotinic acid. Bromine (1.71 g) is added to 10% KOH(30 mL) on ice. The resulting solution is added to finely groundisoquinolinic imide (1.46 g, 9.86 mmol). Upon addition the mixturebegins to foam. When all of the solid is dissolved up aqueous KOH (15%,7 mL) is added and the mixture is heated to 80° C. for 1 min thencooled. The mixture is neutralized with SO₂, and cooled to 0° C. untilprecipitation occurs. The solid is collected by suction filtration andwashed with H₂O, and dried in a vacuum oven to yield of 3-aminoisonicotinic acid (485 mg, 36%) as a white solid. ¹H NMR (DMSO) δ9.5-8.8 (2H, brs), 8.20 (1H, s), 7.70 (1H, d, J=5 Hz), 7.46 (1H, d, J=5Hz).

[0425] 3H-Pyrido[3,4-d]pyrimid-4-one. A mixture of 3-amino isonicotinicacid (485 mg, 3.51 mmol) in formamide (3 mL) is heated to 160° C. for 12h. Upon cooling, the resulting solid is filtered and washed with H₂O anddried in a vacuum oven to yield 3H-pyrido[3,4-d]pyrimid-4-one (373 mg,72%). ¹H NMR (DMSO) δ 12.60 (1H, brs), 9.06 (1H, s), 8.68 (1H, d, J=5.3Hz), 8.23 (1H, s), 7.96 (1H, d, J=5.1 Hz).

[0426] 4-Thiopyrido[3,4-d]pyrimidine. Phosphorous pentasulfide (1.25 g,2.74 mmol) is added to a solution of 3H-pyrido[3,4-d]pyrimid-4-one (366mg, 2.49 mmol) in pyridine (4 mL). The mixture is refluxed for 4 h underN₂. The resulting black tar is dissolved in H20, and a solid forms. Thesolid is filtered and washed with H₂O and dried in a vacuum oven toyield 4-thiopyrido[3,4-d]pyrimidine (369.8 mg, 91%) as a yellow solid.1H NMR (DMSO) δ 14.48 (1H, brs), 9.13 (1H,s), 8.70 (1H, d, J=5.4 Hz),8.29 (1H, s), 8.27 (1H, d, J=5.4 Hz).

[0427] 4-Methylthiopyrido[3,4-d]pyrimidine. A mixture of4-thiopyrido[3,4-d]pyrimidine (369.8 mg, 2.26 mmol), triethylamine (0.6mL, 4.5 mmol), DMSO (2 mL), and iodomethane (0.24 mL, 3.96 mmol) isstirred under N2 at 25° C. for 12 h. The mixture is poured into H₂O andthe resulting solid is filtered and dried in a vacuum oven to yield4-methylthiopyrido[3,4-d]pyrimidine (222 mg, 55%) as a brown solid. ¹HNMR (DMSO) δ 9.51 (1H, s), 9.18 (1H, s), 8.79 (1H, d, J=8 Hz), 7.97 (1H,d, J=8 Hz).

[0428] 4-Anilinopyrido[3.4-d]pyrimidine A mixture of4-methylthiopyrido[3,4-d]pyrimidine (75 mg, 0.42 mmol), and aniline (1mL) is heated to 100° C. under N₂ for 2 h. The reaction mixture is thenchromatographed on silica using MPLC and eluting with a gradient system(CHCl₃ to 5% MeOH in CHCl₃). The fractions are concentrated underreduced pressure, and the resulting solid is recrystallized from Et₂O toyield 4-anilinopyrido[3,4-d]pyrimidine (21.2 mg, 23%) as a yellow solid.¹H NMR (DMSO) δ 10.09 (1H, s), 9.18 (1H, s), 8.74 (1H, d, J=5.3 Hz),8.46 (1H, d, J=5.8 Hz), 7.89 (2H, d, J=8.5 Hz), 7.45 (2H, t, J=7.9 Hz),7.21 (1H, t, J=7.4 Hz).

EXAMPLE 56

[0429] 4-(3-Bromoanilino)pyrido[3,4-d]pyrimidine

[0430] A mixture of 4-methylthiopyrido[3,4-d]pyrimidine (75 mg, 0.42mmol) (see previous experimental), and 3-bromoaniline (1 mL) is heatedto 100° C. under N₂ for 2 h. The reaction mixture is thenchromatographed on silica using MPLC and eluting with a gradient system(CHCl₃ to 5% MeOH in CHCl₃). The fractions are concentrated underreduced-pressure, and the resulting solid is recrystallized from Et₂O toyield 4-(3-bromoanilino)pyrido[3,4-d]pyrimidine (66 mg, 52.7%) as alight brown solid. ¹H NMR (DMSO) δ 10.15 (1H, s), 9.21 (1H, s), 8.80(1H, s), 8.76 (1H, d, J=5.8 Hz), 8.44 (1H, d, J=5.6 Hz), 8.25 (1H, s),7.93 (1H, d, J=7.7 Hz), 7.45-7.37 (2H, m).

EXAMPLE 57

[0431] 4-(3-Bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine

[0432] 5-[N-(tert-Butoxycarbonyl)amino]-2-fluoropyridine.5-Amino-2-fluoropyridine is prepared by hydrogenation (Pd/C) of2-fluoro-5-nitropyridine (obtained from from 2-chloro-5-nitropyridine byreaction with KF in MeCN with Ph₄PBr [J. H. Clark and D. J. Macquarrie,Tetrahedron Lett., 1987, 28, 111-114]. Reaction of the crude amine witht-Boc anhydride gives 5-[N-(tert-butoxycarbonyl)amino]-2-fluoropyridine.¹H NMR (CDCl₃ δ 8.07 (1H, s), 8.05 (1H, m), 6.89 (1H, dd, J=9.2, 3.3Hz), 6.66 (1H, m), 1.52 (9H, s).

[0433] 5-[N-(tert-Butoxycarbonyl)amino]-2-fluoropyridine-4-carboxylicacid. Reaction of 5-[N-(tert-butoxycarbonyl)amino]-2-fluoropyridine (5.3g, 25 mmol) sequentially with n-BuLi and CO₂ as described in thefollowing example gives5-[N-(tert-butoxycarbonyl)amino]-2-fluoropyridine-4-carboxylic acid(1.60 g, 25%). ¹H NMR (DMSO) δ 9.83 (1H, brs), 8.84 (1H, s), 7.49 (1H,d, J=2.9 Hz), 1.47 (9H, s).

[0434] 5-Amino-2-fluoropyridine-4-carboxylic acid. Reaction of5-[N-(tert-butoxycarbonyl)amino]-2-fluoropyridine-4-carboxylic acid (1.0g, 3.9 mmol) with TFA as described above gives5-amino-2-fluoropyridine-4-carboxylic acid (0.46 g, 74%). ¹H NMR (DMSO)δ 7.85 (1H, d, J=1.5 Hz), 7.23 (1H, d, J=2.5 Hz).

[0435] 6-Fluoro-3H-pyrido[3.4-d]pyrimidin-4-one. Reaction of5-amino-2-fluoropyridine-4-carboxylic acid with formamide at 140° C. asabove gave 6-fluoro-3H-pyrido[3,4-d]pyrimidin-4-one (˜20%). ¹H NMR(DMSO) δ 12.48 (1H, m), 8.74 (1H, s), 8.16 (1H, s), 7.63 (1H, d, J=3Hz).

[0436] 4-(3-Bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine. Reaction of6-fluoro-3H-pyrido[3,4-d]pyrimidin-4-one (0.60 g, 3.6 mmol) with POCl₃,followed by reaction of the crude 4,6-dihalo compound with3-bromoaniline gives 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine(0.73 g, 63%). ¹H NMR (DMSO) δ 10.09 (1H, brs), 8.96 (1H, s), 8.75 (1H,s), 8.25 (2H, m), 7.90 (1H, brd, J=6.5 Hz), 7.44-7.34 (2H, m).

EXAMPLE 58

[0437] 4-(3-Bromoanilino)-6-chloropyrido[3,4-d]pyrimidine

[0438] 5-[N-(tert-butoxycarbonyl)-amino]-2-chloropyridine. A mixture of5-amino-2-chloropyridine (12.86 g, 0.1 mol), di-tert-butyldicarbonate(24.0 g, 0.11 mol) and Et₃N (12.1 g, 1.12 mol) in CH₂Cl₂ (150 mL) isheated under reflux for 12 h, cooled, and the precipitate is filteredoff. The organic layer is washed with water, dried (CaCl₂) and filteredthrough a short column of alumina. Removal of the solvent gives5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine (11.9 g, 52%). ¹H NMR(CDCl₃) δ 8.31 (1H, d, J=2.9 Hz), 7.94 (1H, dd, J=8.6, 2.6 Hz), 7.24(1H, d, J=8.7 Hz), 7.15 (1H, m), 1.51 (9 H, s).

[0439] 5-[N-(tert-Butoxycarbonyl)amino]-2-chloropyridine-4-carboxylicacid. A solution of 5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine(22.87 g, 0.1 mol) and TMEDA (47 mL, 0.31 mol) in dry Et₂O (600 mL) iscooled to −78° C., and n-BuLi (10 M in hexanes, 30 mL, 0.3 mol) is addeddropwise. The solution is allowed to warm to −10° C. and is then kept atthat temperature for 2 h, before being recooled to −78° C. Dry CO₂ isthen bubbled in, and the resulting mixture is allowed to warm to 20° C.,before being quenched with water (300 mL) containing a small amount ofNH₄OH. The resulting aqueous layer is washed with EtOAc, then acidifiedslowly with dilute HCl to precipitate5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine-4-carboxylic acid(15.5 g, 57%) ¹H NMR (DMSO) δ 10.00 (1H, s), 9.13 (1H, s), 7.74 (1H, s),1.47 (9H, s).

[0440] 5-Amino-2-chloropyridine-4-carboxylic acid. A stirred suspensionof 5-[N-(tert-butoxycarbonyl)amino]-2-chloropyridine-4-carboxylic acid(1.91 g, 7 mmol) in CH₂Cl₂ (200 mL) is treated slowly withtrifluoroacetic acid until homogeneous (ca. 12 mL). The solution isstirred overnight and extracted with dilute NH₄OH, and the aqueous layeris then acidified with dilute HCl to gave a precipitate5-amino-2-chloropyridine-4-carboxylic acid (1.05 g, 87% yield). ¹H NMR(DMSO) δ 9.01 (2H, m), 8.03 (1H, s) 7.48 (1H, s).

[0441] 6-Chloro-3H-Pyrido[3,4-d]pyrimidin-4-one. A solution of5-amino-2-chloropyridine-4-carboxylic acid (8.1 g, 4.7 mmol) informamide (100 mL) is stirred at 140° C. for 12 h. Dilution of thecooled mixture with water gives a precipitate of6-chloro-3H-pyrido[3,4-d]pyrimidin-4-one (7.3 g, 86% yield). ¹H NMR(DMSO) δ 12.73 (1H, m), 8.90 (1H, d, J=0.7 Hz), 8.23 (1H, s) 7.97 (1H,d, J=0.7 Hz).

[0442] 4,6-Dichloropyrido[3,4-d]pyrimidine. A stirred suspension of6-chloropyrido[3,4-d]pyrimidin-4-one (1.82 g, 10 mmol) in POCl₃ (10 mL)is heated under reflux until dissolved (ca. 2 h) and for a further 30min. Excess reagent is removed under reduced pressure, and the residueis treated with a mixture of CH₂Cl₂ and ice-cold aqueous Na₂CO₃. Theresulting organic layer is dried (Na₂SO₄) and evaporated to give aquantitative yield of crude, unstable,4,6-dichloropyrido[3,4-d]pyrimidine, which is used directly in the nextstep. ¹H NMR (CDCl₃) δ 9.38 (1H, d, J=0.5 Hz), 9.19 (1H, s), 8.09 (1H,d, J=0,5 Hz).

[0443] 4-(3-Bromoanilino)-6-chloropyrido[3,4-d]pyrimidine. A mixture ofthe above crude dichloropyrimidine and 3-bromoaniline (3.8 g, 22 mmol)is dissolved in i-PrOH (100 mL). One drop of conc. HCl is added toinitiate the reaction, and the mixture is then heated under reflux for30 min, cooled, and diluted with water to precipitate4-(3-bromoanilino)-6-chloropyrido[3,4-d]pyrimidine (1.26 g, 38-yield).¹H NMR (DMSO) δ 10.12 (1H, s), 9.03 (1H, s), 8.77 (1H, s), 8.63 (1H, s),8.21 (1H, s), 7.89 (1H, d, J=8.1 Hz), 7.43-7.32 (2H, m).

EXAMPLE 59

[0444] 4-(3-Bromoanilino)-6-methoxypyrido[3,4-d]pyrimidine

[0445] Treatment of 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine(see a previous experimental) at 100 ° C. in a pressure vessel withsodium methoxide in methanol gives4-(3-bromoanilino)-6-methoxypyrido[3,4-d]pyrimidine. ¹H NMR (DMSO) δ9.93 (1H, s), 8.94 (1H, s), 8.61 (1H, s), 8.26 (1H, brs), 7.94 (1H, brd,J=7.6 Hz), 7.88 (1H, s), 7.43-7.32 (2H, m), 4.01 (3H, s).

EXAMPLE 60

[0446] 4-(3-Bromoanilino)-6-methylaminopyrido[3,4-d]pyrimidine

[0447] Treatment of 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine(0.20 g, 0.63 mmol) (see a previous experimental) at 100° C. in apressure vessel with methylamine in ethanol followed by chromatographyon alumina (CH₂Cl₂/MeOH, 99:1) gives4-(3-bromoanilino)-6-methylaminopyrido[3,4-d]pyrimidine (0.07 g, 34%).¹H NMR (DMSO) δ 9.69 (1H, s), 8.75 (1H, s), 8.41 (1H, s), 8.21 (1H,brs), 7.93 (1H, brd, J=7.6 Hz), 7.41-7.28 (2H, m), 7.06 (1H, s), 6.82(1H, q, J=5.0 Hz), 4.95 (3H, d, J=5.0 Hz)

EXAMPLE 61

[0448] 4-(3-Bromoanilino)-6-dimethylaminopyrido[3,4-d]pyrimidine

[0449] Treatment of 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine(see a previous experimental) at 100° C. in a pressure vessel withdimethylamine in ethanol gives4-(3-bromoanilino)-6-dimethylaminopyrido[3,4-d]pyrimidine. ¹H NMR (DMSO)δ 9.71 (1H, s), 8.83 (1H, s), 8.43 (1H, s), 8.21 (1H, brs), 7.94 (1H,brd, J=7.5 Hz), 7.42-7.29 (2H, m), 7.26 (1H, s), 3.17 (6H, s)

EXAMPLE 62

[0450] 4-(Benzylamino)pyrido[3,4-d]pyrimidine

[0451] A mixture of 4-methylthiopyrido[3,4-d]pyrimidine (74 mg, 0.41mmol) (see a previous experimental), and benzylamine (1 mL) is heated to100° C. for 2 h. On cooling the mixture is concentrated under reducedpressure and purified directly by preparative tlc on silica gel elutingwith CH₂Cl₂, to yield 4-(benzylamino)pyrido[3,4-d]pyrimidine (21.2 mg,20%). ¹H NMR (DMSO) δ 9.21 (1H, t, J=5.8 Hz), 9.19 (1H, s), 8.63 (1H, d,J=5.8 Hz), 8.58 (1H, s), 8.20 (1H, d, J=5.1 Hz), 7.41-7.30 (4H, m), 7.26(1H, t, J=7.1 Hz).

EXAMPLE 63

[0452] 4-(3-Bromoanilino)pyrido[2,3-d]pyrimidine

[0453] 3H-pyrido[2,3-d]pyrimidin-4-one. 2-Amino nicotinic acid (15 g,108.6 mmol) in formamide (35 mL) is heated to 165-170° C. for 3.5 h.Upon cooling a solid precipitates. The solid is filtered and washed withH₂O and dried in a vacuum oven to give 3H-pyrido[2,3-d]pyrimidin-4-one(7.87 g, 49.4%). 1H NMR (DMSO) δ 12.50 (1H, s),8.97 (1H, dd, J=1.9, 4.5Hz), 8.53 (1H, dd, J=2.1, 7.9 Hz), 8.34 (1H, s), 7.57 (1H, dd, J=4.6,8.0 Hz).

[0454] 4-Thiopyrido[2,3-d]pyrimidine. Phosphorous pentasulfide (6 g,13.5 mmol) is added to a solution of 3H-pyrido[2,3-d]pyrimidin-4-one (2g, 13.5 mmol) in pyridine (50 mL). The mixture is refluxed for 3 h. Uponcooling a solid formed and the pyridine is decanted off. The solid issuspended in H₂O (20 mL) and then filtered and dried in a vacuum oven toyield 4-thiopyrido[2,3-d]pyrimidine (1.72 g, 78%). ¹H NMR (DMSO)δ 9.06(1H, dd, J=1.9, 4.3 Hz), 8.90 (1H, dd, J=1.9, 8.2 Hz), 8.36 (1 H, s),7.65 (1H, dd, J=4.3, 8.2 Hz).

[0455] 4-Methylthiopyrido[2,3-d]pyrimidine. A mixture of4-thiopyrido[2,3-d]pyrimidine (100 mg, 0.76 mmol), triethylamine (154mg, 1.52 mmol), DMSO (2 mL), and iodomethane (161 mg, 1.14 mmol) isstirred for 12 h at 25° C. The mixture is poured into H₂O and extractedwith EtOAc. The combined extracts are washed with water, saturatedbrine, and dried (MgSO₄), and the solvent is removed under reducedpressure to yield 4-methylthiopyrido[2,3-d]pyrimidine (134 mg, quant.).¹H NMR (DMSO) δ 9.25 (1H, dd, J=1.8, 4.2 Hz), 9.17 (1H, s), 8.59 (1H,dd, J=1.9, 8.2 Hz), 7.75 (1H, dd, J=4.3, 8.2 Hz), 2.73 (3H, s).

[0456] A mixture of 4-methylthiopyrido[2,3-d]pyrimidine (157 mg, 0.89mmol, and 3-bromoaniline (1 mL) is heated to 100° C. for 2 h. On coolinga precipitate forms which is filtered then washed with EtOH and airdried to yield 4-(3-bromoanilino)pyrido[2,3-d]pyrimidine (55.5 mg, 20%.¹H NMR (DMSO) δ 10.13 (1H, s), 9.11 (1H, dd, J=1.7, 4.3 Hz), 9.01 (1H,dd, J=1.7, 8.2 Hz), 8.81 (1H, s), 8.22 (1H, s), 7.90 (1H, d, J=7.7 Hz),7.71 (1H, dd, J=4.3, 8.0 Hz), 7.40 (2H, m).

EXAMPLE 64

[0457] 4-(3-Bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine

[0458] 2,6-Difluoronicotinic acid. 2,6-Difluoropyridine (7.89 mL, 0.087mmol) is added dropwise under N₂ at 78° C. to a stirred solution oflithium diisopropylamide (59.0 mL of a 1.5 N solution in cyclohexane,0.089 mmol) in THF (250 mL). After 2 h at 78° C., a stream of dry CO₂ ispassed through the solution and the mixture is diluted with water andwashed with EtOAc. The aqueous portion is neutralized with 3 N HCl,extracted with EtOAc and worked up to give 2,6-difluoronicotinic acid(13.4 g, 97%). ¹H NMR (DMSO) δ 8.59 (1H, dd, J=9.2, 8.2 Hz), 7.30 (1H,dd, J=8.2, 2.1 Hz), 4.03 (1H, brs).

[0459] 2,6-Difluoronicotinamide. A solution 2,6-difluoronicotinic acid(7.4 g, 0.046 mmol) and SOCl₂ (20 mL) in 1,2-dichloroethane (60 mL)containing DMF (1 drop) is heated under reflux for 4 h, thenconcentrated to dryness under reduced pressure. The residue is dissolvedin Et₂O (100 mL), cooled to 0° C., and treated dropwise withconcentrated ammonia (10.0 mL, 0.17 mmol). After 10 min the solution iswashed with aqueous NaHCO₃ and worked up to give2,6-difluoronicotinamide (5.61 g, 76%). ¹H NMR (CDCl₃) δ 8.70 (1H, dd,J=9.6, 8.3 Hz), 7.00 (1H, ddd, J=8.3, 2.9, 1.1 Hz), 6.71, 6.55 (1H, 1H,2 brs).

[0460] 2-Amino-6-fluoronicotinamide. A solution of2,6-difluoronicotinamide (4.68 g, 0.029 mmol) in dry formamide (30 mL)is saturated with ammonia and allowed to stand at room temperature for24 h. Water (50 mL) is added and the resultant precipitate is filteredoff and washed well with water, to give 6-amino-2-fluoronicotinamide(1.41 g, 31%) mp 236-237° C. ¹H NMR (DMSO) δ 7.89 (1H, dd, J=10.4, 8.4Hz), 7.31, 7.16 (1H, 1H, 2 brs,), 6.93 (2H, brs), 6.36 (1H, dd, J=8.4,2.4 Hz).

[0461] The filtrate and washings are combined and extracted exhaustivelywith EtOAc, and the extract is chromatographed on silica gel.EtOAc/petroleum ether (1:1) elutes forerun, while EtOAc/petroleum ether(2:1) and then EtOAc gives 2-amino-6-fluoronicotinamide (1.57 g, 35%),mp (EtOAc/petroleum ether) 199-200° C. [Rogers, R. B. et al., U.S. Pat.No. 4,383,851, record mp 198-200 C. ¹H NMR (DMSO) δ 8.13 (1H, dd,J=10.4, 8.4 Hz), 7.90, 7.30 (1H, 1H, 2 brs), 7.65 (2H, brs), 6.23 (1H,dd, J=8.4, 2.6 Hz).

[0462] A suspension of 2-amino-6-fluoronicotinamide (0.74 g, 4.77 mmol)in triethyl orthoformate (25 mL) is heated at reflux for 8 h. Aftercooling to room temperature the precipitate is filtered off and washedwell with petroleum ether to give 7-fluoropyrido[2,3-d]pyrimid-4(3H)-one(0.76 g, 96%). ¹H NMR (DMSO) δ 12.75 (1H, brs), 8.66 (1H, dd, J=10.4,8.4 Hz), 8.38 (1H, s), 7.33 (1H, dd, J=8.4, 2.6 Hz).

[0463] 4-(3-Bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine. A suspensionof 7-fluoropyrido(2,3-d]pyrimid-4(3H)-one (0.20 g, 1.21 mmol) in POCl₃(10 mL) is heated under reflux for 2 h. The volatiles are then removedunder reduced pressure, and the residue is partitioned between aqueousNaHCO₃ and EtOAc. The organic extract is worked up to give crude4-chloro-7-fluoropyrido[2,3-d]pyrimidine, which is used directly in thenext reaction. A solution of this product (0.20 g, 1.09 mmol) and3-bromoaniline (0.23 mL, 2.18 mmol) in propan-2-ol (1.0 mL) and THF (10mL) containing a trace of conc. HCl is stirred at 20° C. for 1 h, andthen concentrated to dryness. The residue is dissolved in EtOAc, washedwith aqueous NaHCO₃. and worked up to give an oil, which ischromatographed on silica gel. Elution with EtOAc/petroleum ether (1:5)gives 3-bromoaniline, while EtoAc/petroleum ether (1:1) elutes4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine (0.18 g, 47%), mp(MeOH) 211-213° C. ¹H NMR (DMSO) δ 10.18 (1H, brs), 9.17 (1H, t, J=8.6Hz), 8.80 (1H, s), 8.17 (1H, t, J=1.8 Hz), 7.85 (1H, dt, J_(d)=7.6 Hz,J_(t)=1.8 Hz), 7.53 (1H, dd, J=8.6, 2.7 Hz), 7.41-7.34 (2H, m)

EXAMPLE 65

[0464] 7-Amino-4-(3-bromoanilino)pyrido[2,3-d]pyrimidine

[0465] A solution of 4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine(0.20 g, 0.63 mmol) in EtOH (20 mL) is saturated with ammonia and warmedat 100° C. in a pressure vessel for 30 h. The solvent is removed underreduced pressure to give7-amino-4-(3-bromoanilino)pyrido[2,3-d]pyrimidine (0.18 g, 90%). ¹H NMR(DMSO) δ 9.97 (1H, brs), 8.59 (1H, s), 8.51 (1H, d, J=9.3 Hz), 8.11 (1H,sl brs), 7.77 (1H, brd, J=6.3 Hz), 7.44 (2H, brs), 7.37-7.30 (2H, m),6.81 (1H, d, J=9.3 Hz).

EXAMPLE 66

[0466] 4-(3-Bromoanilino)-7-methylaminopyrido[2,3-d]pyrimidine

[0467] A solution of 4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine(see a previous experimental) (0.20 g, 0.63 mmol), methylaminehydrochloride (0.13 g, 1.88 mmol) and Et₃N (0.30 mL) 2.19 mmol) in EtOH(15 mL) is heated at 100° C. in a pressure vessel for 18 h. The solventis removed under reduced pressure, and the residue is partitionedbetween EtOAc and water. Workup of the organic layer gives4-(3-bromoanilino)-7-(methylamino)pyrido[2,3-d]pyrimidine (0.16 g, 77%).¹H NMR (DMSO) δ 9.53 (1H, s), 8.54 (1H, s), 8.41 (1H, d, J=8.1 Hz), 8.17(1H, t, J=1.8 Hz), 7.83 (1H, dd, J=8.0, 1.9 Hz), 7.66 (1H, brs), 7.32(1H, t, J=8.0 Hz), 7.24 (1H, dd, J=8.0, 1.8 Hz), 6.77 (1H, d, J=8.1 Hz),2.92 (3H, d, J=4.8 Hz).

EXAMPLE 67

[0468] 4-(3-Bromoanilino)-7-dimethylaminopyrido[2,3-d]pyrimidine

[0469] Reaction of 4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine(see a previous experimental) (0.12 g, 0.38 mmol) with dimethylaminehydrochloride (92 mg, 1.13 mmol) and Et₃N (0.18 mL, 1.32 mmol) in EtOH(15 mL) at 100° C. for 18 h in a pressure vessel, followed byevaporation of the solvent and workup, gives4-(3-bromoanilino)-7-(dimethylamino)pyrido[2,3-d] pyrimidine (0.11 g,84%). ¹H NMR (DMSO) δ 9.58 (1H, brs), 8.56 (1H, d, J=9.3 Hz), 8.54 (1H,s), 8.18 (1H, t, J=1.9 Hz), 7.84 (dt, J_(d)=8.0, Hz, J_(t)=1.9 Hz), 7.33(1H, dd, J=8.1, 8.0 Hz) 7.25 (1H, dt, J_(d)=9.3, Hz, J_(t)=1.9 Hz), 7.10(1H, d, J=9.3 Hz), 3.18 (6H, s).

EXAMPLE 68

[0470] 4-(3-Bromoanilino)-7-methoxypyrido[2,3-d]pyrimidine

[0471] A solution of 4-(3-bromoanilino)-7-fluoropyrido[2,3-d]pyrimidine(0.26 g, 0.81 mmol) and sodium methoxide (prepared from 75 mg of sodium,3.26 mmol) in dry MeOH (15 mL) is heated at 90° C. in a pressure vesselfor 18 h. The mixture is poured into water and extracted with EtOAc togive 4-(3-bromoanilino)-7-methoxypyrido[2,3-d]pyrimidine (0.23 g, 86%).¹H NMR (DMSO) δ 9.88 (1H, brs), 8.82 (1H, d, J=8.9 Hz), 8.71 (1H, s),8.18 (1H, dd, J=8.0, 1,9 Hz), 7.36 (1H, dd, J=8.1, 8.0 Hz), 7.29 (1H,ddd, J=8.1, 1.9, 1,9 Hz) 7.15 (1H, d, J=8.9 Hz), 4.01 (3H, s).

EXAMPLE 69

[0472] 4-Benzylamino-7-methylaminopyrimido[4,5-d]pyrimidine

[0473] S-Ethylisothiouronium iodide. A solution of thiourea (3.80 g, 50mmol) and iodoethane (4 mL, 50 mmol) in MeOH (100 mL) is refluxed for 24h. The solvent is stripped under reduced pressure, and the residuallight yellow oil, is dried under vacuum, solidifying spontaneously. Thedesired compound (13.98 g) is obtained quantitatively.

[0474] 4-Amino-5-cyano-2-ethylthiopyrimidine A suspension of NaOMe (2.7g, 50 mmol) in EtOH (200 mL) is added to a mixture of S-ethylisothioureahydroiodide (11.58 g, 50 mmol), ethoxymethylidenemalo-nonitrile (6.1 g,50 mmol) and ethanol (250 mL) at 25° C. The reaction mixture is refluxedunder N₂ for 2 h, and then the solution is concentrated on a hot plateuntil precipitation is observed. After cooling, the solid is collectedby suction filtration and is stirred in water at 25° C. Filtration andvacuum oven drying affords 4-amino-5-cyano-2-ethylthiopyrimidine (4.02g, 45%) as a brown solid. ¹H NMR 6 (DMSO) 8.45 (1H, s), 7.90 (2H, brs),3.00 (2H, q, J=7.3 Hz), 1.27 (3H, t, J=7.3 Hz).

[0475] 4-Amino-2-ethylthiopyrimidine-5-carboxamide.4-Amino-5-cyano-2-ethylthiopyrimidine (4.0 g, 22.3 mmol) is added tosulfuric acid (conc., 4.3 mL) in small portions. The mixture is thenstirred under N₂ at 40° C. for 1.5 h. The reaction is quenched withice-water and NH₄OH is used to adjust the pH to ˜9. The solid iscollected via suction filtration and dried in a vacuum oven overnight.4-Amino-2-ethylthiopyrimidine-5-carboxamide (2.58 g, 58%) is obtained asa light brown solid. 1H NMR: (DMSO) δ 8.52 (1H, s), 7.98 (2H, brs), 7.42(2H, brs), 3.04 (2H, q, J=7.3 Hz), 1.27 (3H, t, J=7.3 Hz).

[0476] 4-Oxo-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine. A mixture of4-amino-2-ethylthiopyrimidine-5-carboxamide (4.66 g, 23.5 mmol) andtriethyl orthoformate (150 mL) is refluxed under N₂ for 24 h, and isthen cooled to 25° C. The brown solid is isolated by suction filtrationand dried in a vacuum oven to give4-oxo-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine (3.54 g, 72%). ¹H NMR:(DMSO) δ 12.80 (1H, s), 9.20 (1H, s), 8.45 (1H, s), 3.18 (2H, q, J=7.4Hz), 1.35 (3H, t, J=7.4 Hz).

[0477] 4-Thiono-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine. A mixture of4-oxo-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine (1.33 g, 6.7 mmol), P₂S₅(1.48 g, 6.6 mmol) and pyridine (15 mL) is refluxed under N₂ for 3 h.The pyridine is then stripped under reduced pressure, and the residue isdissolved in NaOH solution (0.5 M, 75 mL) and boiled with charcoal.After filtration, the filtrate is neutralized with acetic acid togenerate a gold brown solid. Buchner filtration and drying in a vacuumoven affords 4-thiono-7-ethylthio-3H-pyrimido[4,5-d]pyrimidine (1.42 g,95%). ¹H NMR (DMSO) δ 9.47 (1H, s), 8.46 (1H, s), 3.20 (2H, q, J=7.3Hz), 1.35 (3H, t, J=7.3 Hz).

[0478] 7-Ethylthio-4-methylthiopyrimido[4,5-d]pyrimidine. The sameprocedure described for 7-amino-4-methylthiopyrido[4,3-d]pyrimidine inExample 21 is used. ¹H NMR (DMSO) δ 9.52(1H, s), 9.15 (1H, s), 3.23 (2H,q, J=7.3 Hz), 2.72 (3H, s), 1.38 (3H, t, J=7.3 Hz).

[0479] 4-Denzylamino-7-ethylthiopyrimido[4,5-d]pyrimidine. The sameprocedure described for 7-amino-4-anilinopyrido[4,3-d]pyrimidine inexample 21 is used.

[0480] 4-Benzylamino-7-methylaminopyrimido[4,5-d]pyrimidine.4-Benzylamino-7-ethylthiopyrimido[4,5-d]pyrimidine in EtOH containingexcess methylamine is heated to 150° C. in a stainless steel bomb for 5h. The solid is filtered off and dried to give4-benzylamino-7-methylaminopyrimido[4,5-d]pyrimidine.

EXAMPLE 70

[0481] 4-Benzylamino-7-hydrazinopyrimido[4,5-d]pyrimidine

[0482] 4-Benzylamino-7-ethylthio pyrimido[4,5-d]pyrimidine in EtOHcontaining excess hydrazine is heated to 150° C. in a stainless steelbomb for 5h. The solid is filtered off and dried to give4-benzylamino-7-hydrazinopyrimido[4,5-d]pyrimidine.

EXAMPLE 71

[0483] 4-(3-Bromoanilino)thieno[3,2-d]pyrimidine hydrochloride

[0484] 3H-Thieno[3,2-d]pyrimid-4-one. A mixture of methyl3-aminothiophene-2-carboxylate (1 g, 6.3 mmol) and formamide (2 g) isheated at 240° C. for 10 min. Upon cooling a precipitate appeared. It isdissolved in EtOH and filtered. The filtrate is concentrated underreduced pressure and the residue is purified by silica gelchromatography eluting with 10% MeOH in CH₂Cl₂ to yield3H-thieno[3,2-d]pyrimid-4-one (249 mg, 26%) as a solid. ¹H NMR (DMSO) δ12.61 (1H, brs), 8.20 (1H, s), 8.17 (1H, d, J=5 Hz), 7.42 (1H, d, J=5Hz).

[0485] 4-Chlorothieno[3,2-d]pyrimidine. To a solution of DMS (170.3 μL,2.2 mmol) and dichloroethane (1.2 mL) at 0° C. under N_(2,), oxalylchloride (279.2 mg, 3.2 mmol) is added slowly and stirred for 10 min.3H-thieno[3,2-d]pyrimid-4-one (152.2 mg, 1.0 mmol) is added and refluxedfor 5 h. The reaction mixture is poured into water and extracted withCH₂Cl₂. The organic layer is stripped under reduced pressure to yield4-chlorothieno[3,2-d]pyrimidine (140 mg, 82%) as a yellow solid. ¹H NMR(DMSO) δ 9.05 (1H, s), 8.62 (1H, d, J=5 Hz), 7.79 (1H, d, J=5 Hz)

[0486] 4-(3-Bromoanilino)thieno[3,2-d]pyrimidine hydrochloride. Amixture of 4-chlorothieno[3,2-d]-pyrimidine (135 mg, 0.79 mmol) and3-bromoaniline (95 μL, 0.89 mmol) in 2-methoxyethanol (2 mL) is heatedto 79° C. for 30 min. The resulting precipitate is filtered and washedwith CH₂Cl₂ to yield 4-(3-bromo-anilino)thieno[3,2-d]pyrimidinehydrochloride (195.5 mg, 72%) as a light yellow solid. ¹H NMR (DMSO) δ11.33 (1H, s), 8.94 (1H, s), 8.23 (1H, s), 8.53 (1H, d, J=5.3 Hz), 8.07(1H, s), 7.77 (1H, d, J=7.9 Hz), 7.6 (1H, d, J=5.3 Hz), 7.48 (2H, m).

EXAMPLE 72

[0487] 4-Benzylaminothieno[3,2-d]pyrimidine

[0488] As described in the previous experiment4-chlorothieno[3,2-d]pyrimidine (100 mg, 0.586 mmol) and benzylamine(710 μL, 0.645 mmol) in 2-methoxyethanol (2 mL) yields4-benzylaminothieno[3,2-d]pyrimidine (37 mg, 26%). ¹H NMR (DMSO) δ 8.42(1H, s), 8.12 (1H, d, J=5.5 Hz), 7.39 (1H, d, J=5.3 Hz), 7.40-7.30 (4H,m), 7.24 (1H, t, J=6.8 Hz).

EXAMPLE 73

[0489] 4-(3-Bromoanilino)thieno[2,3-d]pyrimidine

[0490] Methyl 2-aminothiophene-3-carboxylate. A mixture of methylcyanoacetate (3.25 g, 32.3 mmol), 1,4 dithiane-2,5 diol (5 g, 32.8mmol), triethylamine (1 mL, 7.71 mmol) in EtOH (50 mL) is stirred at 40°C. for 1 h. The cooled solution is eluted through a silica plug withCH₂Cl₂ . The filtrate is stripped to dryness to give crude methyl2-aminothiophene-3-carboxylate which is carried on to the next reaction.¹H NMR (DMSO) δ 7.26 (1H, s), 6.82 (1H, d, J=5.8 Hz), 6.28 (1H, d, J=5.8Hz), 3.69 (3H, s).

[0491] 3H-Thieno[2,3-d]pyrimid-4-one. A solution of methyl2-aminothiophene-3-carboxylate (602.1 mg, is 3.83 mmol) in formamide (5mL) is heated at 200° C. for 12 h. The resulting tar is dissolved inCH₂Cl₂ (10 mL) then placed on a silica plug and eluted with 10% MeOH inCH₂Cl₂. The filtrate is stripped under reduce pressure and the resultingsolid is washed with EtOH to yield 3H-thieno[2,3-d]pyrimid-4-one (231.4mg, 40%) as an orange solid. ¹H NMR (DMSO) δ 12.50 (1H, brs), 8.13 (1H,s), 7.60 (1H, d, J=5.8 Hz), 7.41 (1H, d, J=6.0 Hz).

[0492] 4-Chlorothieno[2,3-d]pyrimidine. To a solution of DMF (90 μL) andCH₂Cl₂ (2 mL) at 0° C. under N_(2,), oxalyl chloride (148 mg, 1.2 mmol)is added slowly and stirred for 10 min. 3H-Thieno[2,3-d]pyrimid-4-one(81 mg, 0.52 mmol) is added as a solid to the solution and warmed with aheat gun until the solid dissolves. The reaction is stirred at 25° C.for 12 h under N₂. The reaction mixture is poured into water andextracted with CH₂Cl₂ . The phases are separated and the organic layeris dried (Na₂SO₄) and stripped under reduced pressure to yield4-chlorothieno[2,3-d]pyrimidine (87.6 mg, 97%) as a solid. ¹H NMR (DMSO)δ 8.96 (1H, s), 8.17 (1H, d, J=6.0 Hz), 7.62 (1H, d, J=6.0 Hz).

[0493] 4-(3-Bromoanilino)thieno[2,3-d]pyrimidine hydrochloride. Amixture of 4-chlorothieno[2,3-d]pyrimidine (135 mg, 0.79 mmol) and3-bromoaniline (95 μL, 0.89 mmol) in 2-methoxyethanol (2 mL) is heatedto 79° C. for 30 min with stirring. The resulting solid is filtered andwashed with CH₂Cl₂ to yield 4-(3-bromoanilino)thieno[2,3-d]pyrimidinehydrochloride (197 mg, 73%). ¹H NMR (DMSO) δ 9.99 (1H, s), 8.60 (1H, s),8.23 (1H, s), 7.98 (1H, d, J=6.0 Hz), 7.88 (1H, d, J=8.0 Hz), 7.79 (1H,d, J=6.0 Hz), 7.37 (1H, t, J=8.0 Hz), 7.30 (1H, d, J=8.0 Hz).

EXAMPLE 74

[0494] 4-Benzylaminopyrrolo[2 3-d]pyrimidine

[0495] 4-Benzylaminopyrrolo[2,3-d]pyrimidine is prepared as describedpreviously. G. H. Hitchings, K. W. Ledig and R. A. West, U.S. Pat. No.3,037,980, 1962; Chemical Abstracts 1962, 57, 15130c.

EXAMPLE 75

[0496] N⁶-(3-Bromophenyl)adenine

[0497] A mixture of 6-chloropurine (1.0 g, 6.47 mmol), 3-bromoaniline(0.78 mL, 7.12 mmol), and conc HCl (4 drops) in isopropanol (10 mL) isstirred at 80° C. for 5 h. Upon cooling, it precipitates. The solid isfiltered and washed with isopropanol and air dried to yieldN⁶-(3-bromophenyl)adenine (1.93 g, 91%) as a light yellow solid. ¹H NMR(DMSO) δ 11.38 (1H, s), 8.78 (1H, s), 8.75 (1H, s), 7.90 (1H, d, J=8.0Hz), 7.38-7.34 (2H, m)

EXAMPLE 76

[0498] N⁶-Benzyladenine

[0499] N⁶-Benzyladenine is available commercially from the AldrichChemical Company, 1001 West Saint Paul Avenue, Milwaukee, Wis. 53233.

EXAMPLE 77

[0500] 7-Amino-4-(3-methylanilino)pyrido[4.3-d]pyrimidine

[0501] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (217 mg,1.13 mmol) and m-toluidine (1.50 g, 14.0 mmol) is stirred at 155° C. for30 min. The resulting product is chromatographed over silica gel (5%MeOH/CH₂Cl₂) to give 7-amino-4-(3-methylanilino)pyrido[4,3-d]pyrimidine(190 mg, 67%) as a pale yellow solid. ¹H NMR (DMSO) δ 9.81 (1H, brs),9.34 (1H, s), 8.38 (1H, s), 7.60 (2H, s), 7.26 (1H, dd, J=8.5, 7.6 Hz),6.95 (1H, d, J=7.4 Hz), 6.63 (2H, brs), 6.44 (1H, s), 2.33 (3H, s).

EXAMPLE 78

[0502] 7-Amino-4-(4-methoxyanilino)pyrido[4,3-d]pyrimidine

[0503] A mixture of 7-amino-4-methylthiopyrido[4,3-d]pyrimidine (129 mg,0.62 mmol) and 4-methoxyaniline ((0.15 g, 1.2 mmol) was in ethanol (5mL) was heated at 40° C. for 16 h, and then reflux for 3 h. The reactionmixture was cooled to 0° C. overnight, and the solid was colected byvacuum filtration and recrystalized from isopropanol to give7-amino-4-(4-methoxyanilino)pyrido[4,3-d]pyrimidine (42 mg, 25%) as ayellow solid. ¹H NMR (DMSO) δ 10.00 (1H, brs), 9.31 (1H, s), 8.35 (1H,s), 7.62 (2H, d, J=9.2 Hz), 6.96 (2H, d, J=9.2 Hz), 6.70 (2H, slbrs),6.41 (1H, s), 3.77 (3H, s).

EXAMPLE 79

[0504] 4-(3-Bromoanilino)-6-(piperidin-1-yl)pyrido[3,4-d]pyrimidine

[0505] Treatment of 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimidine(see a previous experimental) at 100° C. in a pressure vessel withpiperidine in ethanol gives4-(3-bromoanilino)-6-dimethylaminopyrido[3,4-d]pyrimidine.

[0506] The pharmaceutical compositions of the invention can take any ofa wide variety of oral and parenteral dosage forms. The dosage formscomprise as the active components an inhibitor as defined previously.

[0507] For preparing pharmaceutical compositions, one uses inert,pharmaceutically acceptable carriers that can be either solid or liquid.Solid form preparations include powders, tablets, dispersible granules,capsules, cachets, and suppositories. A solid carrier can be one or moresubstances which may also act as dilutents, flavoring agents,solubilizers, lubricants, suspending agents, binders, or tabletdisintegrating agents; it can also be an encapsulating material. Inpowders, the carrier is a finely divided solid which is in admixturewith the finely divided active compounds. In the tablet, the activecompounds are mixed with carrier having the necessary binding propertiesin suitable proportions and compacted in the shape and size desired. Thepowders and tablets preferably contain from 5% or 10% to about 70% ofactive ingredients. Suitable solid carriers are magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, alow melting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compounds withencapsulating materials as carrier, providing a capsule in which theactive components (with or without other carriers) are surrounded bycarrier, which are thus in association with it. Similarly, cachets areincluded. Tablets, powders, cachets, and capsules can be used as soliddosage forms suitable for oral administration.

[0508] Liquid form preparations include solutions, suspensions, andemulsions. As an example may be mentioned water or water-propyleneglycol solutions for parenteral injection. Liquid preparations can alsobe formulated in solution in aqueous polyethylene glycol solution.Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizing, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive components in water with viscous material, i.e., natural orsynthetic gums, resins, methyl cellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

[0509] Preferably, the pharmaceutical preparation is in unit dosageform. In such form, the preparation may be subdivided into unit dosescontaining appropriate quantities of inhibitor and other anti-cancermaterials individually or as a combination, i.e., in a mixture. The unitdosage form can be a packaged preparation, the package containingdiscrete quantities of preparation, for example, packeted tablets,capsules, and powders in vials or ampoules. The unit dosage form canalso be a capsule, cachet, or tablet itself or it can be the appropriatenumber of any of these in packaged form. Additionally, the unit dosageform may be a dividable form having an inhibitor in one part and otheranti-cancer materials in the other part, such as, a dividable capsule, adividable package, or a two-part ampoule; vial or the like.

[0510] The quantity of an inhibitor in unit dosages of preparation maybe varied or adjusted from about 0.01 mg/kg to 100.0 mg/kg, preferably0.03 mg/kg to less than 1.0 mg/kg of inhibitor.

[0511] The pharmaceutical compositions preferably are constituted sothat they can be administered parenterally or orally. Solutions of theactive compounds as free bases and free acids or pharmaceuticallyacceptable salts can be prepared in water suitable mixed with asurfactant such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0512] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of the microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion, and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, paragens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferred to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions of agents delaying absorption, for example,gelatin.

[0513] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients, into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of thesterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yields a powder of active ingredients plus an additionaldesired ingredient from a previously sterile-filtered solution thereof.

[0514] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active ingredient, its use in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

[0515] It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suitable as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive materials calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the novel dosage unit forms of the invention are dictated by anddirectly dependent on (a) the unique characteristics of the activematerials and the particular therapeutic effect to be achieved, and (b)the limitation inherent in the art of compounding such active materialsfor the treatment of disease in living subjects having a diseasedcondition in which bodily health is impaired as herein disclosed indetail.

[0516] The principal active ingredients are compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in dosage unit form as hereinbeforedisclosed. A unit parenteral dosage form can, for example, contain theprincipal active compound, i.e. an inhibitor, in amounts ranging fromabout 0.5 to about 100 mg, with from about 0.1 to 50 mg being preferred.The daily parenteral doses for mammalian subjects to be treated rangesfrom 0.01 mg/kg to 10 mg/kg of the inhibitor. The preferred daily dosagerange is 0.1 mg/kg to 1.0 mg/kg.

[0517] For oral dosages, the daily amount may range from 0.01 mg ofactive compound/kg of mammalian subject to 100 mg/kg, preferably 0.1 to10 mg/kg of subject.

[0518] The inhibitor described above may form commonly known,pharmaceutically acceptable salts such as alkali metal and other commonbasic salts or acid addition salts, etc. References to the basesubstances are therefore intended to include those common salts known tobe substantially equivalent to the parent compound and hydrates thereof.

[0519] The active compounds described herein are capable of furtherforming both pharmaceutically acceptable acid addition and/or basesalts. All of these forms are within the scope of the present invention.

[0520] Pharmaceutically acceptable acid addition salts of the activecompounds include salts derived from nontoxic inorganic acids such ashydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic,hydrofluoric, phosphorous, and the like, as well as the salts derivedfrom nontoxic organic acids, such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonicacids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate,sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate,oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate,mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate,lactate, maleate, tartrate, methanesulfonate, and the like. Alsocontemplated are salts of amino acids such as arginate and the like andgluconate, galacturonate (see, for example, Berge, S. M. et al,“Pharmaceutical Salts”, JOURNAL OF PHARMACEUTICAL SCIENCE, 66, pp. 1-19(1977)).

[0521] The acid addition salts of said basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. Preferably, anactive compound can be converted to an acidic salt by treating with anaqueous solution of the desired acid, such that the resulting pH is lessthan 4. The solution can be passed through a C18 cartridge to absorb thecompound, washed with copious amounts of water, the compound eluted witha polar organic solvent such as, for example, methanol, acetonitrile,and the like, and isolated by concentrating under reduced pressurefollowed by lyophilization. The free base form may be regenerated bycontacting the salt form with a base and isolating the free base in theconventional manner. The free base forms differ from their respectivesalt forms somewhat in certain physical properties such as solubility inpolar solvents, but otherwise the salts are equivalent to theirrespective free base for purposes of the present invention.

[0522] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for-example, Berge, S. M. et al, “Pharmaceutical Salts”, JOURAL OFPHARMACEUTICAL SCIENCE, 66, pp. 1-19 (1977)).

[0523] The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. Preferably, anactive compound can be converted to a base salt by treating with anaqueous solution of the desired base, such that the resulting pH isgreater than 9. The solution can be passed through a C18 cartridge toabsorb the compound, washed with copious amounts of water, the compoundeluted with a polar organic solvent such as, for example, methanol,acetonitrile and the like, and isolated by concentrating under reducedpressure followed by lyophilization. The free acid form may beregenerated by contacting the salt form with an acid and isolating thefree acid in the conventional manner. The free acid forms differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents, but otherwise the salts are equivalentto their respective free acids for purposes of the present invention.

[0524] Certain of the compounds of the present invention can exist inunsolvated forms as well as solvated forms, including hydrated forms. Ingeneral, the solvated forms, including hydrated forms are equivalent tounsolvated forms and are intended to be encompassed within the scope ofthe present invention.

[0525] Certain of the compounds of the present invention possess one ormore chiral centers and such center may exist in the R(D) or S(L)configuration. The present invention includes all enantiomeric andepimeric forms as well as the appropriate mixtures thereof.

[0526] While the forms of the invention herein constitute presentlypreferred embodiments, many others are possible. It is not intendedherein to mention all of the possible equivalent forms or ramificationsof the invention. It is understood that the terms used herein are merelydescriptive rather than limiting and that various changes may be madewithout departing from the spirit or scope of the invention.

1 1 14 amino acids amino acid linear peptide 1 Lys His Lys Lys Leu AlaGlu Gly Ser Ala Tyr Glu Glu Val 1 5 10

What is claimed is:
 1. A compound of Formula I:

where: two of A-E are nitrogen, with the remaining atom(s) carbon; X isNH or NR⁷, such that R⁷ is lower alkyl (1-4 carbon atoms), OH, NH₂,lower alkoxy (1-4 carbon atoms) or lower monoalkylamino (1-4 carbonatoms); n is 0, 1, 2; R¹ is H or lower alkyl (1-4 carbon atoms); if n is2, R¹ is independently H or lower alkyl (1-4 carbon atoms) on eitherlinking carbon atom; R² is lower alkyl (1-4 carbon atoms), cycloalkyl(3-8 carbon atoms), lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8carbon atoms), nitro, halo (fluoro, chloro, bromo, iodo), lowerperfluoroalkyl (1-4 carbon atoms), hydroxy, lower acyloxy (1-4 carbonatoms; —O—C(O)R), amino, lower mono or dialkylamino (1-4 carbon atoms),lower mono or dicycloalkylamino (3-8 carbon atoms), hydroxymethyl, loweracyl (1-4 carbon atoms; —C(O)R), cyano, lower thioalkyl (1-4 carbonatoms), lower sulfinylalkyl (1-4 carbon atoms), lower sulfonylalkyl (1-4carbon atoms), thiocycloalkyl (3-8 carbon atoms), sulfinylcycloalkyl(3-8 carbon atoms), sulfonylcycloalkyl (3-8 carbon atoms), sulfonamido,lower mono or dialkylsulfonamido (1-4 carbon atoms), mono ordicycloalkylsulfonamido (3-8 carbon atoms), mercapto, carboxy,carboxamido (—C(O)—NH₂), lower mono or dialkylcarboxamido (1-4 carbonatoms), mono or dicycloalkylcarboxamido (3-8 carbon atoms), loweralkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbonatoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbonatoms), lower alkynyl (2-4 carbon atoms), or two R² taken together oncontiguous carbon atoms can form a carbocyclic ring of 5-7 members or amonounsaturated 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino or thiomorpholino ring; and m is0-3; wherein Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl,pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl orquinazolinyl; R³, R⁴l R⁵ and R⁶ are independently, H, lower alkyl (1-4carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy (1-4 carbonatoms), cycloalkoxy (3-8 carbon atoms), hydroxy, lower acyloxy (1-4carbon atoms), amino, lower mono or dialkylamino (1-4 carbon atoms),lower mono or dicycloalkylamino (3-8 carbon atoms), lower alkyl (1-4carbon atoms) or cycloalkyl (3-8 carbon atoms), carbonato (−0C(O)OR)where the R is lower alkyl of 1 to 4 carbon atoms or cycloalkyl of 3-8carbon atoms; or ureido or thioureido or N- or O- linked urethane anyone of which is optionally substituted by mono or di-lower alkyl (1-4carbon atoms) or cycloalkyl (3-8 carbon atoms); or lower thioalkyl (1-4carbon atoms), thiocycloalkyl (3-8 carbon atoms), mercapto, loweralkenyl (2-4 carbon atoms), hydrazino,N′-lower alkylhydrazino (1-4carbon atoms), lower acylamino (1-4 carbon atoms), hydroxyl-amino, lowerO-alkylhydroxylamino (1-4 carbon atoms; or any two of R³, R⁴, R⁵ or R⁶taken together on contiguous carbon atoms form a carbocyclic ring of 5-7members or a monounsaturated ring selected from the group consisting of1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl, furanyl,pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl, diazolanyl,piperazinyl, morpholino and thiomorpholino ring; any lower alkyl groupsubstituent on any of the substituents in R³-R⁶ is optionallysubstituted with one or more groups selected from the group consistingof hydroxy, amino, lower mono alkylamino, lower dialkylamino,N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino,N-thiomorpholino and N-piperazino groups; if any of the substitutentsR¹, R², R³, R⁴, R⁵ or R⁶ contain chiral centers, or in the case of R¹create chiral centers on the linking atoms, then all stereoisomersthereof both separately and as racemic and/or diastereoisomeric mixturesare included; or a pharmaceutical salt or hydrate thereof; with thefurther provisos that when E and B are nitrogen and D and A are carbon,R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹is H or lower alkyl; Ar is aryl unsubstituted or substituted by halo,amino, hydroxy or amino; or n is 1 or 2, R¹ is H or lower alkyl; and Aris a heterocyclic ring, then R⁴ cannot be hydrogen amino, lowermonoalkylamino unsubstituted or substituted by hydroxy, lowermonocycloalkylamino unsubstituted or substituted by hydroxy, lowerdialkylamino unsubstituted or substituted by hydroxy, orheterocyclicalkyl amino; or E and B are nitrogen and D and A are carbon,R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹is H or lower alkyl; Ar is aryl, then R⁴ cannot be lower alkylthio; or Eand B are nitrogen and D and A are carbon, R³ and R⁶ are not present, R⁵is hydrogen, X is NH, n is 1, R¹ is H, Ar is phenyl para substituted bydiethylamino, then R⁴ cannot be amino; or E and B are nitrogen and D andA are carbon, R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is1, R¹ is H, Ar is 2-furanyl, then R⁴ cannot be ethylthio or that atleast one of the group R3 and R5 or R4 and R5 substituents must be otherthen hydrogen, lower alkyl (1-4 carbon atoms) or lower alkoxy (1-4carbon atoms).
 2. The compound of claim 1, wherein: R₇ is a hydrogenatom or an alkyl group, Ar is a phenyl group which is substituted by theradical R₂, wherein R₂ is a fluorine, chlorine, bromine or iodine atom,or a C₁₋₄-alkyl, hydroxyl or C₁₋₄-alkoxy group, cycloalkyl, cycloalkoxygroup, alkenyl or alkynyl, a methyl group which is substituted by 3fluorine atoms, a nitro, amino, alkylamino, dialkylamino,cycloalkylamino, N-alkylcycloalkylamino, arylamino, N-alkylarylaminogroup, R₃ or R⁴ is a mecapto, or hydroxyl group.
 3. The compound ofclaim 1 wherein: R₇ is a hydrogen atom or an alkyl group, Ar is a phenylgroup which is substituted by R₂, wherein R₂ is a fluorine, chlorine,bromine or iodine atom, an alkyl, hydroxyl, alkoxy or cycloalkoxy group,or alkenyl, or alkynyl group, or a methyl group which is substituted by3 fluorine atoms, R₃ or R⁴ is a mercapto, or a hydroxyl, or an aminogroup, a mono lower alkyl amino or dilower alkyl amino or an alkylgroup, which can be substituted by a hydroxyl group.
 4. The compound ofclaim 1 wherein: R₇ is a hydrogen atom or a methyl group, R₂ is afluorine, chlorine, bromine or iodine atom, or an alkyl, alkoxy,cycloalkyl, cycloalkoxy, cyano, methoxycarbonyl, ethoxycarbonyl, ethynylor nitro group, or a methyl group which is substituted by 3 fluorineatoms, or amino, alkylamino, di-alkylamino, alkylcarbonylamino, oralkylsulphonylamino, R₃ or R⁴ is hydroxyl group, or alkoxy group.
 5. Thecompound of claim 1 wherein: R₇ is a hydrogen atom or a methyl group, Aris a naphthyl or a phenyl group which is substituted by the radical R₂,wherein R₂ is a fluorine, chlorine, bromine or iodine atom or an alkyl,trifluoromethyl, alkynyl, alkoxy, cycloalkyl, cyano or nitro group, R₃or R⁴ is hydroxyl or cycloalkoxy, or amino group, or a monoalkyl aminogroup.
 6. A pharmaceutical composition adapted for administration as aninhibitor of the epidermal growth factor receptor family of tyrosinekinases, comprising a therapeutically effective amount of a compound ofFormula I in admixture with a pharmaceutically acceptable excipient,diluent or carrier:

where: two of A-E are nitrogen, with the remaining atom(s) carbon; X isNH or NR⁷, wherein R⁷ is lower alkyl (1-4 carbon atoms), OH, NH₂, loweralkoxy (1-4 carbon atoms) or lower monoalkylamino (1-4 carbon atoms); nis 0, 1, 2; R¹ is H or lower alkyl (1-4 carbon atoms); if n=2, R¹ isindependently H or lower alkyl (1-4 carbon atoms) on either linkingcarbon atom; R² is lower alkyl (1-4 carbon atoms), cycloalkyl (3-8carbon atoms), lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8 carbonatoms), nitro, halo (fluoro, chloro, bromo, iodo), lower perfluoroalkyl(1-4 carbon atoms), hydroxy, lower acyloxy (1-4 carbon atoms; —O—C(O)R),amino, lower mono or dialkylamino (1-4 carbon atoms), lower mono ordicycloalkylamino (3-8 carbon atoms), hydroxymethyl, lower acyl (1-4carbon atoms; —C(O)R), cyano, lower thioalkyl (1-4 carbon atoms), lowersulfinylalkyl (1-4 carbon atoms), lower sulfonylalkyl (1-4 carbonatoms), thiocycloalkyl (3-8 carbon atoms), sulfinylcycloalkyl (3-8carbon atoms), sulfonylcycloalkyl (3-8 carbon atoms), sulfonamido, lowermono or dialkylsulfonamido (1-4 carbon atoms), mono ordicycloalkylsulfonamido (3-8 carbon atoms), mercapto, carboxy,carboxamido (—C(O)—NH₂), lower mono or dialkylcarboxamido (1-4 carbonatoms), mono or dicycloalkylcarboxamido (3-8 carbon atoms), loweralkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbonatoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbonatoms), lower alkynyl (2-4 carbon atoms), or two R² taken together oncontiguous carbon atoms can form a carbocyclic ring of 5-7 members or amonounsaturated 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino or thiomorpholino ring; and m is0-3; wherein Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl,pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl orquinazolinyl; R³, R^(4,) R⁵ and R⁶ are independently, H, lower alkyl(1-4 carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy (1-4carbon atoms), cycloalkoxy (3-8 carbon atoms), hydroxy, lower acyloxy(1-4 carbon atoms), amino, lower mono or dialkylamino (1-4 carbonatoms), lower mono or dicycloalkylamino (3-8 carbon atoms), lower alkyl(1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms), carbonato(−0C(O)OR) where the R is lower alkyl of 1 to 4 carbon atoms orcycloalkyl of 3-8 carbon atoms; or ureido or thioureido or N- or O-linked urethane any one of which is optionally substituted by mono ordi-lower alkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms); orlower thioalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbon atoms),mercapto, lower alkenyl (2-4 carbon atoms), hydrazino,N′-loweralkylhydrazino (1-4 carbon atoms), lower acylamino (1-4 carbon atoms),hydroxyl-amino, lower O-alkylhydroxylamino (1-4 carbon atoms); or anytwo of R³, R⁴, R⁵ or R⁶ taken together on contiguous carbon atoms form acarbocyclic ring of 5-7 members or a monounsaturated ring selected fromthe group consisting of 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl,pyranyl, furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl,thiazolanyl, diazolanyl, piperazinyl, morpholino and thiomorpholinoring; any lower alkyl group substituent on any of the substituents inR³-R⁶ is optionally substituted with one or more groups selected fromthe group consisting of hydroxy, amino, lower monoalkylamino, lowerdialkylamino, N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino,N-thiomorpholino and N-piperazino groups; if any of the substitutentsR¹, R², R³, R^(4,)R⁵ or R⁶ contain chiral centers, or in the case of R¹create chiral centers on the linking atoms, then all stereoisomersthereof both separately and as racemic and/or diastereoisomeric mixturesare included; or a pharmaceutical salt or hydrate thereof; with thefurther provisos that when E and B are nitrogen and D and A are carbon,R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹is H or lower alkyl; Ar is aryl unsubstituted or substituted by halo,amino, hydroxy or amino; or n is 1 or 2, R¹ is H or lower alkyl; and Aris a heterocyclic ring, then R⁴ cannot be hydrogen amino, lowermonoalkylamino unsubstituted or substituted by hydroxy, lowermonocycloalkylamino unsubstituted or substituted by hydroxy, lowerdialkylamino unsubstituted or substituted by hydroxy, orheterocyclicalkyl amino; or E and B are nitrogen and D and A are carbon,R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹is H or lower alkyl; Ar is aryl, then R⁴ cannot be lower alkylthio; or Eand B are nitrogen and D and A are carbon, R³ and R⁶ are not present, R⁵is hydrogen, X is NH, n is 1, R¹ is H, Ar is phenyl para substituted bydiethylamino, then R⁴ cannot be amino; or E and B are nitrogen and D andA are carbon, R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is1, R¹ is H, Ar is 2-furanyl, then R⁴ cannot be ethylthio or that atleast one of the group R3 and R5 or R4 and R5 substituents must be otherthen hydrogen, lower alkyl (1-4 carbon atoms) or lower alkoxy (1-4carbon atoms).
 7. The compound of claim 1 wherein R₃ is an amino.
 8. Thecomposition of claim 6 wherein R₃ is an amino.
 9. A compound of FormulaI:

where: A and E are nitrogen, with the remaining atom(s) carbon; X is NHor NR⁷, such that R⁷ is lower alkyl (1-4 carbon atoms), OH, NH₂, loweralkoxy (1-4 carbon atoms) or lower monoalkylamino (1-4 carbon atoms); nis 1, 1, 2; R¹ is H or lower alkyl (1-4 carbon atoms); if n is 2, R¹ isindependently H or lower alkyl (1-4 carbon atoms) on either linkingcarbon atom; R² is lower alkyl (1-4 carbon atoms), cycloalkyl (3-8carbon atoms), lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8 carbonatoms), nitro, halo (fluoro, chloro, bromo, iodo), lower perfluoroalkyl(1-4 carbon atoms), hydroxy, lower acyloxy (1-4 carbon atoms; —O—C(O)R),amino, lower mono or dialkylamino (1-4 carbon atoms), lower mono ordicycloalkylamino (3-8 carbon atoms), hydroxymethyl, lower acyl (1-4carbon atoms; —C(O)R), cyano, lower thioalkyl (1-4 carbon atoms), lowersulfinylalkyl (1-4 carbon atoms), lower sulfonylalkyl (1-4 carbonatoms), thiocycloalkyl (3-8 carbon atoms), sulfinylcycloalkyl (3-8carbon atoms), sulfonylcycloalkyl (3-8 carbon atoms), sulfonamido, lowermono or dialkylsulfonamido (1-4 carbon atoms), mono ordicycloalkylsulfonamido (3-8 carbon atoms), mercapto, carboxy,carboxamido (—C(O)—NH₂), lower mono or dialkylcarboxamido (1-4 carbon 10atoms), mono or dicycloalkylcarboxamido (3-8 carbon atoms), loweralkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbonatoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbonatoms), lower alkynyl (2-4 carbon atoms), or two R² taken together oncontiguous carbon atoms can form a carbocyclic ring of 5-7 members or amonounsaturated 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino or thiomorpholino ring; and m is0-3; wherein Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl,pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl orquinazolinyl; R³, R⁴ R⁵ and R⁶ are independently, not present, H, loweralkyl (1-4 carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy(1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms), hydroxy, loweracyloxy (1-4 carbon atoms), amino, lower mono or dialkylamino (1-4carbon atoms), lower mono or dicycloalkylamino (3-8 carbon atoms), loweralkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms), carbonato(−0C(O)OR) where the R is lower alkyl of 1 to 4 carbon atoms orcycloalkyl of 3-8 carbon atoms; or ureido or thioureido or N- or O-linked urethane any one of which is optionally substituted by mono ordi-lower alkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms); orlower thioalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbon atoms),mercapto, lower alkenyl (2-4 carbon atoms), hydrazino,N′-loweralkylhydrazino (1-4 carbon atoms), lower acylamino (1-4 carbon atoms),hydroxyl-amino, lower O-alkylhydroxylamino (1-4 carbon atoms; or any twoof R³-R⁶ taken together on contiguous carbon atoms form a carbocyclicring of 5-7 members or a monounsaturated ring selected from the groupconsisting of 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino and thiomorpholino ring; any loweralkyl group substituent on any of the substituents in R³-R⁶ isoptionally substituted with one or more groups selected from the groupconsisting of hydroxy, amino, lower mono alkylamino, lower dialkylamino,N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino,N-thiomorpholino and N-piperazino groups; if any of the substitutentsR¹, R², R³, R⁴, R⁵ or R⁶ contain chiral centers, or in the case of R¹create chiral centers on the linking atoms, then all stereoisomersthereof both separately and as racemic and/or diastereoisomeric mixturesare included; or a pharmaceutical salt or hydrate thereof.
 10. Acompound of Formula I:

where: B and E are nitrogen, with the remaining atom(s) carbon; X is NHor NR⁷, such that R⁷ is lower alkyl (1-4 carbon atoms), OH, NH₂, loweralkoxy (1-4 carbon atoms) or lower monoalkylamino (1-4 carbon atoms); nis 0, 1, 2; R¹ is H or lower alkyl (1-4 carbon atoms); if n is 2, R¹ isindependently H or lower alkyl (1-4 carbon atoms) on either linkingcarbon atom; R² is lower alkyl (1-4 carbon atoms), cycloalkyl (3-8carbon atoms), lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8 carbonatoms), nitro, halo (fluoro, chloro, bromo, iodo), lower perfluoroalkyl(1-4 carbon atoms), hydroxy, lower acyloxy (1-4 carbon atoms; —O—C(O)R),amino, lower mono or dialkylamino (1-4 carbon atoms), lower mono ordicycloalkylamino (3-8 carbon atoms), hydroxymethyl, lower acyl (1-4carbon atoms; —C(O)R), cyano, lower thioalkyl (1-4 carbon atoms), lowersulfinylalkyl (1-4 carbon atoms), lower sulfonylalkyl (1-4 carbonatoms), thiocycloalkyl (3-8 carbon atoms), sulfinylcycloalkyl (3-8carbon atoms), sulfonylcycloalkyl (3-8 carbon atoms), sulfonamido, lowermono or dialkylsulfonamido (1-4 carbon atoms), mono ordicycloalkylsulfonamido (3-8 carbon atoms), mercapto, carboxy,carboxamido (—C(O)—NH₂), lower mono or dialkylcarboxamido (1-4 carbonatoms), mono or dicycloalkylcarboxamido (3-8 carbon atoms), loweralkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbonatoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbonatoms), lower alkynyl (2-4 carbon atoms), or two R² taken together oncontiguous carbon atoms can form a carbocyclic ring of 5-7 members or amonounsaturated 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino or thiomorpholino ring; and m is0-3; wherein Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl,pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl orquinazolinyl; R³, R⁴ R⁵ and R⁶ are independently, not present, H, loweralkyl (1-4 carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy(1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms), hydroxy, loweracyloxy (1-4 carbon atoms), amino, lower mono or dialkylamino (1-4carbon atoms), lower mono or dicycloalkylamino (3-8 carbon atoms), loweralkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms), carbonato(−0C(O)OR) where the R is lower alkyl of 1 to 4 carbon atoms orcycloalkyl of 3-8 carbon atoms; or ureido or thioureido or N- or O-linked urethane any one of which is optionally substituted by mono ordi-lower alkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms); orlower thioalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbon atoms),mercapto, lower alkenyl (2-4 carbon atoms), hydrazino,N′-loweralkylhydrazino (1-4 carbon atoms), lower acylamino (1-4 carbon atoms),hydroxyl-amino, lower O-alkylhydroxylamino (1-4 carbon atoms; or any twoof R³-R⁶ taken together on contiguous carbon atoms form a carbocyclicring of 5-7 members or a monounsaturated ring selected from the groupconsisting of 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino and thiomorpholino ring; any loweralkyl group substituent on any of the substituents in R³-R⁶ isoptionally substituted with one or more groups selected from the groupconsisting of hydroxy, amino, lower mono alkylamino, lower dialkylamino,N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino,N-thiomorpholino and N-piperazino groups; if any of the substituents R¹,R², R³, R⁴, R⁵ or R⁶ contain chiral centers, or in the case of R¹ createchiral centers on the linking atoms, then all stereoisomers thereof bothseparately and as racemic and/or diastereoisomeric mixtures areincluded; or a pharmaceutical salt or hydrate thereof; with the furtherprovisos that when E and B are nitrogen and D and A are carbon, R³ andR⁶ are not present, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹ is H orlower alkyl; Ar is aryl unsubstituted or substituted by halo, amino,hydroxy or amino; or n is 1 or 2, R¹ is H or lower alkyl; and Ar is aheterocyclic ring, then R⁴ cannot be hydrogen amino, lowermonoalkylamino unsubstituted or substituted by hydroxy, lowermonocycloalkylamino unsubstituted or substituted by hydroxy, lowerdialkylamino unsubstituted or substituted by hydroxy, orheterocyclicalkyl amino; or E and B are nitrogen and D and A are carbon,R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹is H or lower alkyl; Ar is aryl, then R⁴ cannot be lower alkylthio; or Eand B are nitrogen and D and A are carbon, R³ and R⁶ are not present, R⁵is hydrogen, X is NH, n is 1, R¹ is H, Ar is phenyl para substituted bydiethylamino, then R⁴ cannot be amino; or E and B are nitrogen and D andA are carbon, R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is1, R¹ is H, Ar is 2-furanyl, then R⁴ cannot be ethylthio or that atleast one of the group R3 and R5 or R4 and R5 substituents must be otherthen hydrogen, lower alkyl (1-4 carbon atoms) or lower alkoxy (1-4carbon atoms).
 11. A pharmaceutical composition adapted foradministration as an inhibitor of the epidermal growth factor receptorfamily of tyrosine kinases, comprising a therapeutically effectiveamount of a compound of Formula I in admixture with a pharmaceuticallyacceptable excipient, diluent or carrier:

where: at least two of A-E, namely A and E are nitrogen, with theremaining atom(s) carbon; X is NH or NR⁷, wherein R⁷ is lower alkyl (1-4carbon atoms), OH, NH₂, lower alkoxy (1-4 carbon atoms) or lowermonoalkylamino (1-4 carbon atoms); n is 0, 1, 2; R¹ is H or lower alkyl(1-4 carbon atoms); if n=2, R¹ is independently H or lower alkyl (1-4carbon atoms) on either linking carbon atom; R² is lower alkyl (1-4carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy (1-4 carbonatoms), cycloalkoxy (3-8 carbon atoms), nitro, halo (fluoro, chloro,bromo, iodo), lower perfluoroalkyl (1-4 carbon atoms), hydroxy, loweracyloxy (1-4 carbon atoms; —O—C(O)R), amino, lower mono or dialkylamino(1-4 carbon atoms), lower mono or dicycloalkylamino (3-8 carbon atoms),hydroxymethyl, lower acyl (1-4 carbon atoms; —C(O)R), cyano, lowerthioalkyl (1-4 carbon atoms), lower sulfinylalkyl (1-4 carbon atoms),lower sulfonylalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbonatoms), sulfinylcycloalkyl (3-8 carbon atoms), sulfonylcycloalkyl (3-8carbon atoms), sulfonamido, lower mono or dialkylsulfonamido (1-4 carbonatoms), mono or dicycloalkylsulfonamido (3-8 carbon atoms), mercapto,carboxy, carboxamido (—C(O)—NH₂), lower mono or dialkylcarboxamido (1-4carbon atoms), mono or dicycloalkylcarboxamido (3-8 carbon atoms), loweralkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbonatoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbonatoms), lower alkynyl (2-4 carbon atoms), or two R² taken together oncontiguous carbon atoms can form a carbocyclic ring of 5-7 members or amonounsaturated 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino or thiomorpholino ring; and m is0-3; wherein Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl,pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl orquinazolinyl; R³, R⁴, R⁵ and R⁶ are independently, not present, H, loweralkyl (1-4 carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy(1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms), hydroxy, loweracyloxy (1-4 carbon atoms), amino, lower mono or dialkylamino (1-4carbon atoms), lower mono or dicycloalkylamino (3-8 carbon atoms), loweralkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms), carbonato(−0C(O)OR) where the R is lower alkyl of 1 to 4 carbon atoms orcycloalkyl of 3-8 carbon atoms; or ureido or thioureido or N- or O-linked urethane any one of which is optionally substituted by mono ordi-lower alkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms); orlower thioalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbon atoms),mercapto, lower alkenyl (2-4 carbon atoms), hydrazino,N′-loweralkylhydrazino (1-4 carbon atoms), lower acylamino (1-4 carbon atoms),hydroxyl-amino, lower O-alkylhydroxylamino (1-4 carbon atoms); or anytwo of R³-R⁶ taken together on contiguous carbon atoms form acarbocyclic ring of 5-7 members or a monounsaturated ring selected fromthe group consisting of 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl,pyranyl, furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl,thiazolanyl, diazolanyl, piperazinyl, morpholino and thiomorpholinoring; any lower alkyl group substituent on any of the substituents inR³-R⁶ is optionally substituted with one or more groups selected fromthe group consisting of hydroxy, amino, lower monoalkylamino, lowerdialkylamino, N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino,N-thiomorpholino and N-piperazino groups; if any of the substituents R¹,R², R³, R⁴, R⁵ or R⁶ contain chiral centers, or in the case of R¹ createchiral centers on the linking atoms, then all stereoisomers thereof bothseparately and as racemic and/or diastereoisomeric mixtures areincluded; or a pharmaceutical salt or hydrate thereof; with the furtherprovisos that when E and B are nitrogen and D and A are carbon, R³ andR⁶ are not present, R⁵is hydrogen, X is NH, n is 0, 1 or 2, R¹ is H orlower alkyl; Ar is aryl unsubstituted or substituted by halo, amino,hydroxy or amino; or n is 1 or 2, R¹ is H or lower alkyl; and Ar is aheterocyclic ring, then R⁴ cannot be hydrogen amino, lowermonoalkylamino unsubstituted or substituted by hydroxy, lowermonocycloalkylamino unsubstituted or substituted by hydroxy, lowerdialkylamino unsubstituted or substituted by hydroxy, orheterocyclicalkyl amino; or E and B are nitrogen and D and A are carbon,R³ and R⁶ are not present, R⁵is hydrogen, X is NH, n is 0, 1 or 2, R¹ isH or lower alkyl; Ar is aryl, then R⁴ cannot be lower alkylthio; or Eand B are nitrogen and D and A are carbon, R³ and R⁶ are not present,R⁵is hydrogen, X is NH, n is 1, R¹ is H, Ar is phenyl para substitutedby diethylamino, then R⁴ cannot be amino; or E and B are nitrogen and Dand A are carbon, R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, nis 1, R¹ is H, Ar is 2-furanyl, then R⁴ cannot be ethylthio or that atleast one of the group R3 and R5 or R4 and R5 substituents must be otherthen hydrogen, lower alkyl (1-4 carbon atoms) or lower alkoxy (1-4carbon atoms).
 12. A pharmaceutical composition adapted foradministration as an inhibitor of the epidermal growth factor receptorfamily of tyrosine kinases, comprising a therapeutically effectiveamount of a compound of Formula I in admixture with a pharmaceuticallyacceptable excipient, diluent or carrier:

where: B and E are nitrogen, with the remaining atom(s) carbon; X is NHor NR⁷, wherein R⁷ is lower alkyl (1-4 carbon atoms), OH, NH₂, loweralkoxy (1-4 carbon atoms) or lower monoalkylamino (1-4 carbon atoms); nis 0, 1, 2; R¹ is H or lower alkyl (1-4 carbon atoms); if n=2, R¹ isindependently H or lower alkyl (1-4 carbon atoms) on either linkingcarbon atom; R is lower alkyl (1-4 carbon atoms), cycloalkyl (3-8 carbonatoms), lower alkoxy (1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms),nitro, halo (fluoro, chloro, bromo, iodo), lower perfluoroalkyl (1-4carbon atoms), hydroxy, lower acyloxy (1-4 carbon atoms; —O—C(O)R),amino, lower mono or dialkylamino (1-4 carbon atoms), lower mono ordicycloalkylamino (3-8 carbon atoms), hydroxymethyl, lower acyl (1-4carbon atoms; —C(O)R), cyano, lower thioalkyl (1-4 carbon atoms), lowersulfinylalkyl (1-4 carbon atoms), lower sulfonylalkyl (1-4 carbonatoms), thiocycloalkyl (3-8 carbon atoms), sulfinylcycloalkyl (3-8carbon atoms), sulfonylcycloalkyl (3-8 carbon atoms), sulfonamido, lowermono or dialkylsulfonamido (1-4 carbon atoms), mono ordicycloalkylsulfonamido (3-8 carbon atoms), mercapto, carboxy,carboxamido (—C(O)—NH₂), lower mono or dialkylcarboxamido (1-4 carbonatoms), mono or dicycloalkylcarboxamido (3-8 carbon atoms), loweralkoxycarbonyl (1-4 carbon atoms), cycloalkoxycarbonyl (3-8 carbonatoms), lower alkenyl (2-4 carbon atoms), cycloalkenyl (4-8 carbonatoms), lower alkynyl (2-4 carbon atoms), or two R² taken together oncontiguous carbon atoms can form a carbocyclic ring of 5-7 members or amonounsaturated 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl, pyranyl,furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl, thiazolanyl,diazolanyl, piperazinyl, morpholino or thiomorpholino ring; and m is0-3; wherein Ar is phenyl, thienyl, furanyl, pyrrolyl, pyridyl,pyrimidyl, imidazoyl, pyrazinyl, oxazolyl, thiazolyl, naphthyl,benzothienyl, benzofuranyl, indolyl, quinolinyl, isoquinolinyl orquinazolinyl; R³, R⁴, R⁵ and R⁶ are independently, not present, H, loweralkyl (1-4 carbon atoms), cycloalkyl (3-8 carbon atoms), lower alkoxy(1-4 carbon atoms), cycloalkoxy (3-8 carbon atoms), hydroxy, loweracyloxy (1-4 carbon atoms), amino, lower mono or dialkylamino (1-4carbon atoms), lower mono or dicycloalkylamino (3-8 carbon atoms), loweralkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms), carbonato(−0C(O)OR) where the R is lower alkyl of 1 to 4 carbon atoms orcycloalkyl of 3-8 carbon atoms; or ureido or thioureido or N- orO-linked urethane any one of which is optionally substituted by mono ordi-lower alkyl (1-4 carbon atoms) or cycloalkyl (3-8 carbon atoms); orlower thioalkyl (1-4 carbon atoms), thiocycloalkyl (3-8 carbon atoms),mercapto, lower alkenyl (2-4 carbon atoms), hydrazino,N′-loweralkylhydrazino (1-4 carbon atoms), lower acylamino (1-4 carbon atoms),hydroxyl-amino, lower O-alkylhydroxylamino (1-4 carbon atoms); or anytwo of R³-R⁶ taken together on contiguous carbon atoms form acarbocyclic ring of 5-7 members or a monounsaturated ring selected fromthe group consisting of 1,3-dioxolanyl, 1,4-dioxanyl, 1,4-dioxepinyl,pyranyl, furanyl, pyrrolidyl, piperidinyl, thiolanyl, oxazolanyl,thiazolanyl, diazolanyl, piperazinyl, morpholino and thiomorpholinoring; any lower alkyl group substituent on any of the substituents inR³-R⁶ is optionally substituted with one or more groups selected fromthe group consisting of hydroxy, amino, lower monoalkylamino, lowerdialkylamino, N-pyrrolidyl, N-piperidinyl, N-pyridinium, N-morpholino,N-thiomorpholino and N-piperazino groups; if any of the substituents R¹,R², R³, R⁴, R⁵ or R⁶ contain chiral centers, or in the case of R¹ createchiral centers on the linking atoms, then all stereoisomers thereof bothseparately and as racemic and/or diastereoisomeric mixtures areincluded; or a pharmaceutical salt or hydrate thereof; with the furtherprovisos that when E and B are nitrogen and D and A are carbon, R³ andR⁶ are not present, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹ is H orlower alkyl; Ar is aryl unsubstituted or substituted by halo, amino,hydroxy or amino; or n is 1 or 2, R¹ is H or lower alkyl; and Ar is aheterocyclic ring, then R⁴ cannot be hydrogen amino, lowermonoalkylamino unsubstituted or substituted by hydroxy, lowermonocycloalkylamino unsubstituted or substituted by hydroxy, lowerdialkylamino unsubstituted or substituted by hydroxy, orheterocyclicalkyl amino; or E and B are nitrogen and D and A are carbon,R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is 0, 1 or 2, R¹is H or lower alkyl; Ar is aryl, then R⁴ cannot be lower alkylthio; or Eand B are nitrogen and D and A are carbon, R³ and R⁶ are not present, R⁵is hydrogen, X is NH, n is 1, R¹ is H, Ar is phenyl para substituted bydiethylamino, then R⁴ cannot be amino; or E and B are nitrogen and D andA are carbon, R³ and R⁶ are not present, R⁵ is hydrogen, X is NH, n is1, R¹ is H, Ar is 2-furanyl, then R⁴ cannot be ethylthio or that atleast one of the group R3 and R5 or R4 and R5 substituents must be otherthen hydrogen, lower alkyl (1-4 carbon atoms) or lower alkoxy (1-4carbon atoms).